In situ immunization

ABSTRACT

The arming of activated T cells (ATC) with BiAbs can overcome major barriers for successful adoptive immunotherapy. The BiAb approach takes the advantage of the targeting specificity of monoclonal antibodies and the cytotoxic capacity of T cells to lyse tumors. Arming of ATC with BiAb makes every T cell an antigen-specific CTL and infusions of such cells will markedly increase the effective precursor frequency of CTL in the cancer patient. Furthermore, the ability of such armed ATC to kill multiple times without rearming with BiAb, secrete tumoricidal cytokines, secrete chemokines, and survive in patients for up to 8 days after the last infusion or in Beige/SCID mice for over 13 weeks after cessation of treatment. The persistence of cells in the Beige/SCID after infusion show long-term survival capability in the host. Re-stimulation of armed ATC after 3 cycles of cytotoxicity with tumor cells resulted in the secretion of interferon gamma indicating the development of tumor specific immune responses in the population of cells that have been exposed multiple times to antigen. In summary, armed ATC can act as a cytotoxic “drug”, kill multiple times (direct killing), divide after killing (increasing the effector:target ratio in vivo), secrete tumoricidal cytokines (indirectly killing), secrete chemokines at the tumor site (recruit naive T cells and antigen-presenting cells to immunize the patient to tumor lysate) and persist in patients and animal models for weeks to months (long-term survival).

This application claims priority to U.S. Provisional Application Ser.No. 60/313,164 filed Aug. 17, 2001, the entirety of which isincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides for compositions and methods for a T-cellbased immunotherapy for malignancies or other disease characterized byabnormal cellular proliferation. In particular, the invention relates toin vivo activated T cells armed with chemically heteroconjugatedbispecific monoclonal antibodies generated against tumor antigens. Theinvention provides for treatment using autologous, relatedHLA-identical, partially-related HLA-mismatched, or allogeneic T cellsfrom patients diagnosed with such malignancies such as breast cancer,prostate cancer, renal tumors, or other malignancies, and the generationof antigen-specific long-term memory T cells. The advantages of thepresent invention are that antibodies can be generated against tumorspecific antigens of choice, and the method uses the patient'sautologous, activated T cells armed with the targeting specificity ofthe bispecific antibody for combating the tumor.

2. Background

With early detection, most malignancies can be treated by conventionalsurgery, chemotherapy, or radiotherapy. In contrast, it is nearlyimpossible for the immune system to reject bulky or metastatic disease.The challenge then is to identify antigen specific or non-specificsystems that will improve clinical responses in the treatment ofadvanced cancers and hematologic malignancies.

The key challenge in immunotherapy is to induce the immune system of acancer patient to make a specific immune response to autologous tumors.A few tumor-specific antigens, such as HER-2/neu, malignant melanoma,and p53 are well-characterized and are known to induce in vitro and invivo specific immune responses. Although adoptively transferred T cellscan eliminate or reduce lethal tumor burdens in animals, adapting thisprinciple in humans has been problematic. Moreover, while dramaticclinical responses have been observed in some patients with renal cellcarcinoma and malignant melanoma who received treatment with tumorinfiltrating lymphocytes, the success of murine models did not translateinto higher cure rates in large trials of patients with renal carcinomaand malignant melanoma.

Previous approaches taken included expansion of tumor infiltratinglymphocytes (TIL) that display cytotoxic activity directed at autologoustumor antigens using IL-2, and reinfusion of the TIL into patients withrenal carcinoma (RCC) and metastatic melanoma (MM). TIL are CD3⁺ cellsthat display activated natural killer cell (ANK) activity, but are moreeffective killers than ANK on a per cell basis.¹⁹ TIL have been reportedto traffic to metastatic melanoma lesions.²⁰ Trials using TIL and highdose IL-2 in patients with advanced RCC, MM, and other advanced tumorshave obtained clinical responses ranging from 13 to 60% with mostreports ranging between 15-20%.²²⁻²⁵ The higher responses may be due todifferences in patient selection as well as laboratory processingdifferences.

However, this therapeutic approach has major drawbacks. One limitationof TIL therapy are the toxicities related to high dose IL-2 infusionswhich restrict the use of IL-2 in patients who have poor performancestatus.²⁶⁻²⁸ The major toxicities of IL-2 are fluid gain and capillaryleak leading to respiratory distress and hypotension often requiringvasopressor support and ICU monitoring.²⁶ Other side effects includefever, chills, malaise, diarrhea, increased creatinine, mental statuschanges, cardiac arrhythmias, and rashes.^(27;28) Although high doses ofTIL alone can be infused without toxicities,²⁹ the efficacy of TIL isthought to be linked to co-administration of high dose IL-2. Subsequentstudies suggest that high dose IL-2 alone is equivalent to high doseIL-2 in combination with TIL therapy.

Another drawback to this approach is that the rate of positive clinicalresponses from the combination of TIL and high dose IL-2 is stillunacceptably low. Unfortunately, the anti-tumor activity exhibited byTIL has not been a consistent observation in larger clinical series.¹⁹Therefore, new approaches to generate tumor specific CTL and methods tospecifically target tumors are needed to improve clinical responses.

There is a need in the art to provide for immunotherapeutic approachesthat need to include strategies that address the above issues and otherissues.

SUMMARY OF THE INVENTION

The present invention combines the cytotoxic capability of T cells andthe specificity of antibodies to augment the cytotoxic capacity of Tcells to lyse tumor cells. The present invention further providescompositions and methods for treatment of tumors on an individualpatient basis by arming activated autologous T cells with bispecificantibodies specific for a certain tumor antigen. If the tumor antigenchanges in a patient, the bispecific antibody used to arm the activatedT cell can be replaced with a bispecific antibody that is specific forthe new antigen. Furthermore, long term antigen-specific T cells aregenerated by the methods and compositions disclosed in the presentinvention.

The present invention is advantageous in that the arming of T cells withbispecific antibodies significantly increases the chances of overcomingsome of the major barriers for successful adoptive immunotherapy, suchas for example, tumor escape. This approach, increases the precursorfrequency of CTL directed to specific tumors, and improves specificbinding and enrichment of effector cells at the tumor site, as well asaugmenting tumoricidal activity.

In general the invention provides for the arming of activated T cells(ATC) with a bispecific antibody (BiAb) which targets a tumor antigen.The present approach is advantageous in that it combines the specificityof the antibody directed at CD3 (T cell receptors, TCR) and a tumorantigen to augment the cytotoxic capacity of T cells to lyse, forexample, Her2⁺ prostate tumors. In this illustrative example, the BiAbbridge between the ATC and the Her2⁺ target redirects the cytotoxicityof T cells to the Her2+ targets, while bypassing majorhistocompatibility restrictions. The present invention bypasses thetoxic side effects of co-administered chemokines, such as IL-2, as ATCfrom normal subjects and cancer patients are first grown in low doseIL-2 (100 IU/ml) and then armed with the BiAb, for example,anti-CD3×anti-Her2 (Her2Bi). These ATC target and kill Her2⁺ breast,prostate, and pancreatic carcinoma cell lines. The invention alsoprovides for immunotherapy (IT) comprising multiple infusions ofbispecific antibody armed ATC, IL-2, and granulocyte-macrophage colonystimulating factor (GM-CSF).

Another major advantage of the present invention is that only nanogramamounts of bispecific antibodies are needed to produce the desiredcytotoxic effect, because the cytotoxicity is very specifically directedand localized towards the tumor. Other advantages of the presentinvention are the use of autologous patient cells, thereby, avoiding anygraft-versus-host-reaction complications; reinfused cells can be washedso that there in no carry over of antibodies or cytokines; andbispecific antibodies can be produced against an emergent new tumorantigen during the progression of the disease.

In a preferred embodiment, a patient suffering from cancer is treatedaccording to the method of the invention which comprises the steps ofisolating the patient's peripheral blood mononuclear cells. Thepatient's T cells are activated by ex vivo stimulation with eithersoluble anti-CD3 monoclonal antibody, or anti-CD3 and anti-CD28monoclonal antibodies attached to a solid support. The activated T cellsare expanded in the presence of about 100 IU/ml of IL-2. Once a suitablenumber of activated T cells is achieved, for example, between about 1-10billion activated T cells, the T cells are armed with bispecificantibodies. The bispecific antibodies are capable of binding to the Tcell receptor complex of a T cell, to tumor-associated antigens on atumor cell, and to Fc-receptors of accessory cells via the Fc part ofthe antibody. The cytotoxic activity is tested in vitro against tumorcells and an appropriate arming dose of bispecific antibodies isdetermined, based on the cytotoxic activity. The patient is thenreinfused with a composition of the autologous cells, which comprisesthe activated T cells armed with a bispecific antibody, immunocompetentnaïve or mature T cells, immunocompetent naïve or mature B cells, anddendritic cells.

In another preferred embodiment the bispecific antibody is comprised oftwo monoclonal antibodies, chemically heteroconjugated to form thebispecific antibody, preferably in a 1:1 ratio. The preferredspecificity of the bispecific antibody is for tumor antigens, forexample Her2⁺ and the T cell receptor complex. Most preferred T cellreceptor antigens are the CD3 and CD28. In one aspect of the invention,the monoclonal antibodies forming the bispecific antibody are humanizedmonoclonal antibodies or are genetically engineered, using methods wellknown to one of skill in the art.

In another preferred embodiment, the autologous, activated T cells arearmed with at least about 0.5 ng antibody per million T cells, morepreferably at least about 10 ng antibody per million T cells, mostpreferably to at least about 100 ng antibody per million T cells. Thearming dose, however, is optimized for each individual patient bytitrating a frozen aliquot of the patients activated T cells to achievea percent specific cytotoxicity level at an effector to target ratio of25:1 of at least about 30% against a tumor target.

In another preferred embodiment, the infusing dose of armed T cells ispreferably about 2 billion, more preferably about 10 billion, mostpreferably at least about 40 billion armed T cells. In one aspect of theinvention, the patient receives at least about four infusions, morepreferably about six infusions, most preferably at least about 10infusions.

In another preferred embodiment the activated T cell is either CD3/CD8positive and/or a CD3/CD4 positive cell. In one aspect of the inventionthe armed T cells from a patient can be co-administered with other formsof therapy. The autologous T cells can be transduced with vectors codingfor chemokines thereby producing a high concentration of localized ofchemokines. The invention is thus, also suitable for treatingimmunosuppressed patients.

In another preferred embodiment, patients which have been treated withATC or COACT, induces immunological memory to the specific antigenrecognized by the infused T cells. The memory T cells preferably exhibita mature or secondary immune response so that the immune response ismore rapid and more specific as compared to a naïve or primary immuneresponse. Memory cells are easily identifiable, e.g. by flow cytometricanalysis.

In another preferred embodiment, the ATC or COACT are able to carry outmultiple cycles of tumor cell killing. That is, the same cell, cantarget and kill tumor cells more than once after infusion into thepatient.

In another preferred embodiment, multiple exposure of the anti-CD3activated polyclonal T cell population induces the development ofantigen specific T cell clones, for example HER2/neu. Preferably, theinduction of antigen specific clones directed at a specific tumorantigen allows for the maturation of the cellular response so as toinduce the production of T cells that recognize antigens on the tumorthat are yet undefined and unknown.

In another preferred embodiment, the ATC or COACT are infused into thepatient in the absence of antigen presenting cells, for example,dendritic cells.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating the steps for heteroconjugating OKT3with the anti-tumor associated antigen MAb (anti-TAA) (9184).

FIG. 2 shows the analysis of the heteroconjugation product on anon-reducing SDS gel electrophoresis using a 2-15% gradient.

FIG. 3 shows an overnight culture, whereby, rosetting of ATC armed withHer2Bi with MCF-7 or PC-3 cells is shown.

FIG. 4 A-E shows a histogram illustrating the binding of IgG1 (9184portion of Her2Bi) to SK-BR-3 and MCF-7 cells, respectively.

FIG. 5 is a graph showing the development of specific cytotoxicitydirected at MCF-7 by armed ATC, fresh PBMC or ATC from normal subjects.

FIG. 6 shows composite titration curves for unarmed ATC and ATC armedwith 0.5, 5.0, and 50.0 ng of Her2Bi at E/T between 5 and 25. Each curverepresents the interpolated mean % (±SEM) specific cytotoxicity of 3experiments directed at MCF-7 targets.

FIG. 7 shows a the results from a cytotoxic assay illustrating thecytotoxic activity of enriched T cell subsets to determine the T cellsubsets for specific cytotoxicity.

FIG. 8 is a bar graph showing how long ATC remain armed and continue tokill tumor cells.

FIG. 9 shows the results of a cytotoxic assay from the fractionresponsible for binding and targeting CEA. The dimer and multimercontaining fractions are separated from the monomer fractions using aSephacryl 300 column and testing each fraction after adjusting allprotein concentrations to the same for % specific cytotoxicity directedat LS174T.

FIG. 10 are graphs which summarize data from 10 normal subjects armedwith 50 ng of OKT3×9184 per 10⁶ ATC.

FIG. 11 are graphs showing in vivo ATC mediated cytotoxicity. ATC fromcancer patients that had been cryopreserved using control rate freezingon the day of culture indicated on each panel, were thawed and armedwith Her2Bi. ATC were tested for cytotoxicity directed at MCF-7.

FIG. 12 shows a bar graph illustrating cytokine secretion induced bybinding of ATC to SK-BR-3 tumor cells.

FIG. 13 shows a graph illustrating in vitro cytotoxicity mediated byarmed ATC or COACTS with Her2Bi against various tumor HER2⁺ tumortargets.

FIG. 14 is a graph showing increasing arming doses of OKT3×9184,increases the % specific cytotoxicity directed at PC-3, by armed ATC.

FIG. 15 shows flow cytometry results obtained from Her2BiAb binding toPC-3, MCF-7 and SK-BR-3 cells.

FIG. 16 is a gel showing the results of the chemical conjugation ofOKT3×Herc, using the scheme in FIG. 1. BCA protein is quantitated andanalyzed by non-reducing SDS gel electrophoresis using a 2-15% gradientgel and Coomassie blue staining is used to visualize proteins in thegel.

FIG. 17 is a graph showing the ability of ATC from two normal subjectsto lyse SK-BR-3 targets.

FIG. 18 is a graph showing the comparative arming doses betweenOKT3×9184 and OKT3×Herc.

FIG. 19 is a bar graph showing interferon-y secretion by armed ATC after24 hrs of culture with target cells.

FIG. 20 is a graph showing the comparison of cytolytic capabilities ofHer2Bi or OKT3×Herc armed normal ATC using PC-3 targets.

FIG. 21 is a graph showing that armed patient T cells remain cytotoxicto PC-3 after freeze/thawing.

FIG. 22 is a bar graph showing IFNγ ELISPOTS produced by normal ATCarmed with OKT3×9184 upon binding with PC-3 cells. FIG. 22 summarizesdata as spots/million armed ATC.

FIG. 23 is a graph illustrating that cytotoxicity mediated by ATC armedwith OKT3×Herc is not inhibited by soluble Herceptin®.

FIG. 24 is a graph illustrating that ATC armed with old and new lots ofOKT3×9184, are stable for at least 11 months.

FIG. 25 is a graph illustrating that subcutaneous coinjections of armedATC (20×10⁶) and CEA+LS174 (1×10⁶) colon carcinoma cell line (WinnAssay) prevented tumor progression and death in 40% of the mice thatreceived armed ATC whereas only 10% of the mice that receive ATC alonesurvived more than 100 days.

FIG. 26 is a bar graph illustrating cytokine secretion by normal ATCarmed with Her2Bi or OKT3×Herc induced by binding to PC-3.

FIG. 27 is a bar graph illustrating cytokine secretion by armed T cellsfrom patients who have received multiple cycles of chemotherapy.

FIGS. 28 and 29 are graphs illustrating the clinical results of armingdoses needed for specific cytotoxicity. FIG. 28 shows the data for thenormal (NL) and FIG. 29 shows the data for the patient (PT).

FIG. 30 is a graph illustrating that cryopreservation had little affecton ATC specific cytotoxicity directed at Her2+ MCF-7 targets.

FIG. 31 is a graph illustrating the cytotoxic ability of T cells byarming of anti-CD3/anti-CD28 coactivated T cells (COACTS) with Her2Bi(OKT3×9184 or OKT3×Herc).

FIG. 32 is a graph illustrating the clinical data using cancer patients'COACTS and ATC armed with OKT3×9184. COACTS and ATC have comparablespecific cytotoxicity activity against MCF-7 targets. Panels P1, P2, andP3 show the ability of unarmed and armed COACTS and ATC to mediatespecific cytotoxicity.

FIG. 33 is a graph showing specific cytotoxicity for one normal subjectand two patients with prostate cancer at effector to target ratios from3.13 to 25.00.

FIG. 34 is a graph showing that armed ATC were able to kill MIA targets,a pancreatic cell line.

FIG. 35 is a graph showing the 1^(st) cytotoxicity assay mediated by analiquot of armed T cells and an aliquot of unarmed ATC tested at timezero. After 48 hrs of incubation with the first set of targets, theunarmed and armed ATC were harvested and aliquots of each were replatedonto a second set of targets for a second culture and 2^(nd)cytotoxicity assay at 45 hrs. After the replated unarmed (ATC) or armedATC (aATC) were co-cultured with SK-BR-3 between 48 hrs and 96 hrs, theunarmed and armed ATC were harvested and aliquots of each were replatedonto a third set of targets for a third culture and a 3^(rd)cytotoxicity assay at 96 hrs. Finally, the unarmed and armed ATC thathad been co-cultured with SK-BR-3 from 96 hrs to 215 hrs were harvestedand aliquots of each were replated onto a fourth set of targets in a4^(th) cytotoxicity assay at 215 hrs.

FIG. 36 are results from a flow cytometry assay showing the numbers ofCFDA-SE+cells within the CD4 or CD8 subsets that had been armed withOKT3×Herceptin, OKT3×Rituxan, or left unarmed. CD4+ population showedevidence of cell division with a very distinct population of cells thathad divided and showed reduce intensity whereas the CD4+ cells armedwith OKT3×Rituxan and unarmed ATC did not show as many cells that haddivided and exhibited half as much staining intensity.

FIG. 37 are graphs from flow cytometry data showing survival ortrafficking in patients by detecting IgG2a bearing armed ATC.

FIG. 38 is a graph showing Percent specific cytotoxicity toward PC-3tumor targets is elevated through the arming of activated T-cells withBiAb. Cytotoxicity is measured here with three E/T ratios of 6.25, 12.5,and 25:1. The % standard error of the mean (SEM) is depicted with errorbars at each E/T ratio. Unarmed ATC () kill less than half of all tumortargets at the highest E/T, while ATC armed with 5 ng (□), 50 ng (▴),and 500 ng (▪) are capable of almost 100% tumor cell lysis at a 25:1E/T.

FIG. 39 is a graph showing A comparison of unarmed ( ) armed (▪), andirrelevant BiAb armed ATC reveals that armed ATC exhibit markedlyelevated levels of cytokine and chemokine secretion over the controls.Unarmed ATC cytokine secretion of TNF-α and IFN-γ was too minimal forrepresentation in this chart. T-cells and PC-3 targets were plated at a10:1 E/T ratio and incubated overnight at 37° C.

FIG. 40 is a Kaplan-Meier plot showing The tumor-free survivalproportion is greatest among mice given the highest dose (2×10⁷) ofarmed ATC (orange line). Survival is improved above the rate of thecontrol (◯) first in mice receiving the lower dose (10⁷) of armed ATC(▴), then in mice receiving the same dose (10⁷) of unarmed ATC (▪).

FIG. 41 is a graph showing tumor growth delay curves depicting efficacyof unarmed and armed ATC in preventing PC-3 tumors. The highest dose ofarmed ATC (2×10⁷) (▴) is completely effective in delaying tumor growthby clay 105, whereas treatment with 10⁷ armed ATC will not prevent butcan delay tumor growth (Δ). Although not as effective as the equivalentdose of armed ATC in delaying tumor growth, 10⁷ unarmed ATC (♦) alsodelayed tumor growth above the rate of the control (▪).

FIG. 42 is a graph showing tumor growth delay. The highest dose (5×10⁷)of armed ATC plus IL-2 (▴) delays tumor growth out to day 90 whencompared with the IL-2 control group (▪), p=0.0636. The low dose of 10⁷aATC (Δ) is not effective in delaying tumor growth when compared withcontrol mice.

FIG. 43 is a Kaplan-Meier plot showing that sufficient numbers of armedATC induce long remissions in mice with established tumors. Tumorsdisappeared after 6 weeks of treatment in 2 of 5 mice (40%) given 5×10⁷armed ATC plus IL-2, twice per week (Δ). Tumors disappeared in 1 of 6mice (17%) after 6 weeks of treatment with 10⁷ armed ATC plus IL-2 (□).All control mice (given IL-2 alone) were sacrificed due to tumor burdenwithin six weeks of primary injection with tumor targets (◯). Treatmentwith armed ATC plus IL-2 or IL-2 alone was not initiated untilestablished tumors reached 5×5 mm in dimension.

FIG. 43 is a bar graph showing the number of IFN gamma ELISPOTS from ATC(unarmed activated T cells that had been exposed 3 times to SK-BR-3),unarmed ATC that were exposed to a human EBV-driven B cell lines (a Bcell line would not express HER2/neu receptors; only the final time),and aATC (armed ATC that were exposed to SK-BR-3 three times and thenexposed a fourth time in this assay). The assay was performed on day 20after arming. No additional arming was performed from the initial armingwith 50 ng/million.

DETAILED DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTSTHEREOF

The present invention provides novel compositions and methods for thetreatment of tumors. The compositions comprise activated T cells armedwith bispecific antibodies specifically capable of binding to tumorantigens ultimately resulting in the destruction of the tumor. Thepresent invention is advantageous in that it uses a patient's own Tcells, activates the patient's T cells ex vivo and arms the T cells withantibodies specific for the patients tumor. The reinfusion of thepatients own activated and armed T cells with a composition comprisingnaïve T and B cells as well as powerful antigen presenting cells such asdendritic cells provides for an increase in the patients own precursorcytotoxic cellular pool of cells and induction of long-term memory Tcells.

Before the present modified T-cells, compositions and methods oftreatment are described, it is to be understood that this invention isnot limited to particular cell lines, excipients or method stepsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aT-cell” includes a plurality of such T-cells and reference to “theantibodies” includes reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

In general, the present invention provides for a “drug” which iscomprised of anti-CD3 activated T cells (ATC) or anti-CD3 and anti-CD28co-activated T cells (COACTS) armed with chemically heteroconjugatedanti-CD3 (OKT3)×anti-Her-2/neu (9184) or anti-CD3 (OKT3)×anti-Her2/neu(Herceptin) bispecific monoclonal antibodies (BiAb). ATC are produced byex vivo stimulation with soluble OKT3 (about 10-20 ng/ml) and culturedin at least about 100 to about 600 IU/ml IL-2. COACTS are produced bycoactivation with anti-CD3 (OKT3)/anti-CD28 (9.3) monoclonal antibodiesco-immobilized on beads. ATC or COACTS are armed with pretitrated dosesof bispecific antibodies (BiAb) based on functional cytotoxicitydirected at Her-2⁺ tumor cells. The non-binding monoclonal antibodies orheteroconjugates are washed away from the armed ATC or COACTS.

ATC or COACTS armed with BiAb will be interchangeably referred to as“the drug” for the purposes of this disclosure. This drug providesspecific anti-tumor effect for patients who have Her2/neu+ tumors suchas breast, renal, prostate, and other HER2 tumors. However, this antigenis merely an illustrative example and is not meant to be construed aslimiting in any way. Any antibody raised against an individual patientstumor may be used. This has the major advantage of monitoring anyantigenic changes of the tumor, allowing for tailoring treatment on anindividual or disease progression stage. The other major advantage ofthe present invention is that neither ATC or COACTS have causeddose-limiting cell-based toxicities. Furthermore, no monoclonalantibodies are infused in patients thereby, removing any risk of toxicside-effects.

In a preferred embodiment, the ATC or COACTs undergo multiple cycles oftumor antigen recognition and tumor cell killing. The ability of theseATC or COACTs to kill multiple cells is shown in the examples whichfollow. Assays used to identify the ATC and/or COACTs and determinetheir ability to kill tumor cells is discussed in great detail infra andin the examples which follow. For example, an armed T cell which istargeted to tumor antigens such as, for example, Her2⁺ tumors, can beidentified by labeling the T cell with a fluorescent marker, orsecondary antibody that is detectable by flow cytometry or other methodswell-known to one of ordinary skill in the art. Examples of labels fordetection of the armed T cells include but not limited to, greenfluorescent proteins, avidins and the like. As an illustrative example,biopsies from a tumor to which tumor specific T cells armed withbispecific antibodies and labeled with a detectable marker, are used toconfirm the presence of the armed T cells at the site of the tumor. Thebiopsied tissue is processed by methods well known in the art andprepared for use in cell detection assays.

In another preferred embodiment, multiple exposure of the anti-CD3activated polyclonal T cell population induces the development ofantigen specific T cell clones, for example HER2/neu. As an illustrativeexample which is not meant to be construed or limiting in any way, thedata shown in FIG. 44 show the number of IFN gamma ELISPOTS from ATC(unarmed activated T cells that had been exposed 3 times to SK-BR-3),unarmed ATC that were exposed to a human EBV-driven B cell lines (a Bcell line would not express HER2/neu receptors; only the final time),and aATC (armed ATC that were exposed to SK-BR-3 three times and thenexposed a fourth time in this assay). The assay was performed on day 20after arming. No additional arming was performed from the initial armingwith 50 ng/million. Without wishing to be bound by theory,subpopulations of armed ATC were primed to HER2/neu and have becomememory cells as measured by their ability to respond vigorously torechallenge to HER2/neu antigen on the SK-BR-3 cells. Furthermore, theresults suggest that multiple exposure of the anti-CD3 activatedpolyclonal T cell population has selected or induced the development ofHER2/neu specific T cell clones.

Preferably, the induction of antigen specific clones directed at aspecific tumor antigen allows for the maturation of the cellularresponse so as to induce the production of T cells that recognizeantigens on the tumor that are yet undefined and unknown.

In another preferred embodiment, the ATC or COACT are infused into thepatient without any antigen presenting cells, for example, dendriticcells. As discussed in the Examples which follow, the armed T cells areadministered to a patient without any dendritic cells in the culturesystem. Without wishing to be bound by theory, one very real possibilityis that activated T cells can act as professional antigen presentingcells since they upregulate class II upon activation and may acttogether with the crosslinked tumor antigen in the presence of cytokineand chemokines produced by the reactivation process to induce antigenspecific CTL. Class II upregulation may provide the necessary help fromCD4 helper cells in the polyclonal mixture to provide the signals neededto induce antigen-specific CTL.

In another preferred embodiment, the present invention has the advantageof activating “by-stander” T cells, not just specifically one particularstimulating antigen, thus a bigger immune response is produced leadingto the production of more lymphokines and subsequently greaterimmunoglobulin production by B cells.

Another advantage of the present invention is the maintenance of theperipheral pool of memory T cells (CD45RO⁺) as memory T cells can beexpanded (proliferated) without the need of specific antigenicstimulation to maintain the clonal size. Also the naive T cellrepertoire (CD45RA⁺) can be maintained, as the present invention allowsthe proliferation of naive T cells and/or precursor cells. For example,to evaluate the frequency of resting T cells with memory phenotype thatcould be stimulated by cytokines to grow, limiting dilution experimentscan be performed. CD45RO⁺ CD4⁺ resting T cells can be cultured with IL-2alone or in combination with TNF-α and IL-6, in the presence ofautologous irradiated macrophages and anti-DR antibodies to preventautoreactive responses.

A “precursor cell” can be any cell in a cell differentiation pathwaythat is capable of differentiating into a more mature cell. As such, theterm “precursor cell population” refers to a group of cells capable ofdeveloping into a more mature cell. A precursor cell population cancomprise cells that are totipotent, cells that are pluripotent and cellsthat are stem cell lineage restricted (i.e. cells capable of developinginto less than all hematopoietic lineages, or into, for example, onlycells of erythroid lineage). As used herein, the term “totipotent cell”refers to a cell capable of developing into all lineages of cells.Similarly, the term “totipotent population of cells” refers to acomposition of cells capable of developing into all lineages of cells.Also as used herein, the term “pluripotent cell” refers to a cellcapable of developing into a variety (albeit not all) lineages and areat least able to develop into all hematopoietic lineages (e.g.,lymphoid, erythroid, and thrombocytic lineages). For example, apluripotent cell can differ from a totipotent cell by having the abilityto develop into all cell lineages except endothelial cells. A“pluripotent population of cells” refers to a composition of cellscapable of developing into less than all lineages of cells but at leastinto all hematopoietic lineages. As such, a totipotent cell orcomposition of cells is less developed than a pluripotent cell orcompositions of cells. As used herein, the terms “develop”,“differentiate” and “mature” all refer to the progression of a cell fromthe stage of having the potential to differentiate into at least twodifferent cellular lineages to becoming a specialized cell. Such termscan be used interchangeably for the purposes of the present application.

As used herein, the term “population” refers to cells having the same ordifferent identifying characteristics. The term “lineage” refers to allof the stages of the development of a cell type, from the earliestprecursor cell to a completely mature cell (i.e. a specialized cell).Systemic memory T cells are characterized according to the cell surfaceexpression of certain known antigens. Typically these cells are positivefor CD4, and lack expression of CD45RA, and integrin α4β7. They arefurther characterized by expression of CCR4. A subset of cells ofinterest are common leukocyte antigen positive (CLA⁺). Verification ofthe identity of the cells of interest may be performed by any convenientmethod, including antibody staining and analysis by fluorescencedetection, ELISA, etc., reverse transcriptase PCR, transcriptionalamplification and hybridization to nucleic acid microarrays, etc.

Some memory T cells associated with the skin are known to express CLA,and such cells are of particular interest for treatment with the presentmethods, particularly to modulate the trafficking, or homing of thesecells to cutaneous tissues, for treatment, for example of melanomas.

Other systemic memory cells are triggered to adhere to endothelialICAM-1, by LFA-1 binding. These adhesion molecules are implicateddirectly in graft rejection, psoriasis, and arthritis. A CCR4 blockingagent that prevents triggering of LFA-1 mediated adhesion is useful inthe inhibition of graft rejection by preventing the accumulation ofmemory T cells at the site of graft implantation; preventing intra-isletinfiltration by T cells to inhibit development of insulin-dependentdiabetes mellitus; blocking infiltration of T cells into the centralnervous system to treat multiple sclerosis and other demyelinatingdiseases; blocking the accumulation of T cells in the synovial joints ofpatients suffering from rheumatoid arthritis; accumulation of memory Tcells to influence immune responsiveness, and the like. Thus, the drug(ATC, COACT) of the invention allows for treatments of diseases otherthan tumors as the bispecific antibody can be specific for any desiredepitope.

In accordance with the invention, T cells from patients are, preferablyactivated ex vivo either by soluble anti-CD3 antibody, or areco-activated by using anti-CD3 and anti-CD28 monoclonal antibodies,either by soluble or immobilized on a solid support. A preferred solidsupport are plastics, or any surface upon which antibodies can beimmobilized, or beads, such as, for example, Dynal beads. Onceactivated, T cells are armed with a bispecific antibody. The locationand movements through the patient's body of these activated T cells canbe monitored by using a labeled antibody that binds to a desiredmolecule on the surface of the activated T cell or directed to a portionof the bispecific antibody, such as for example, the F_(c) region.Monitoring of the cells is achieved by using the flow cytometry methodsof the invention. The T cells can also be labeled by agents which aredetectable by any imaging techniques known in the art.

By “patient” herein is meant a mammalian subject to be treated, withhuman patients being preferred. In some cases, the methods of theinvention find use in experimental animals, in veterinary application,and in the development of animal models for disease, including, but notlimited to, rodents including mice, rats, and hamsters; and primates.

The terms “treatment”, “treating”, and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure of a disease and/oradverse effect attributed to the disease. In general, methods of theinvention involve treating diseases generally referred to as cancer andmay be applied to a variety of different types of cancers by utilizingantibodies which specifically bind antigens known to be present on thesurfaces of cancer cells of the type of cancer being treated.“Treatment” as used herein covers any treatment of such a disease in amammal, particularly a human, and includes:

-   -   (a) Preventing and/or diagnosing the disease in a subject which        may be predisposed to the disease but has not yet been diagnosed        as having it;    -   (b) Inhibiting the disease, i.e. arresting it's development;        and/or    -   (c) Relieving the disease, i.e. causing regression of the        disease.

The invention is directed towards treating patients with cancer and isparticularly directed towards treating types of cancer which are notgenerally treatable by normal surgical methodologies. More specifically,“treatment” is intended to mean providing a therapeutically detectableand beneficial effect on a patient suffering from cancer. That effectcan include stimulating the patient's own immune system to aid intreating the cancer.

Although a variety of types of cancer can be treated the antibodiesattached to the T-cells will generally determine the type of cancerwhich can be treated. The antibodies must have a sufficiently high levelof binding affinity to antigens on the cancer cells which arc beingtargeted. As used herein in order to consider the antibodies to be“specific” or have a sufficiently high binding affinity, e.g. theantibodies will have a binding affinity of about 10⁻⁷ moles/liter, orabout 10⁻⁸ to about 10⁹ moles/liter and may be up to 10⁻¹¹ moles/literor higher for the epitope of interest which is preferably specific tothe cell surface of the cancer being targeted. It will be understood bythose skilled in the art that the term “specific” as used in connectionwith binding affinity refers to such a high affinity binding, and is notintended to mean that the binding affinity can not bind to othermolecules as well. One may find cross-reactivity with differentepitopes, e.g. to relatedness of an antigen sequence for structure, orthe structure of the antibody binding pocket itself. Antibodiesdemonstrating such cross-reactivity are still considered specific forthe purposes of the present invention.

“Immune cells” as used herein, is meant to include any cells of theimmune system that may be assayed, including, but not limited to, Blymphocytes, also called B cells, T lymphocytes, also called T cells,natural killer (NK) cells, lymphokine-activated killer (LAK) cells,monocytes, macrophages, neutrophils, granulocytes, mast cells,platelets, Langerhans cells, stem cells, dendritic cells, peripheralblood mononuclear cells, tumor-infiltrating (TIL) cells, gene modifiedimmune cells including hybridomas, drug modified immune cells, andderivatives, precursors or progenitors of the above cell types.

“Activity”, “activation” or “augmentation” is the ability of immunecells to respond and exhibit, on a measurable level, an immune function.Measuring the degree of activation refers to a quantitative assessmentof the capacity of immune cells to express enhanced activity whenfurther stimulated as a result of prior activation. The enhancedcapacity may result from biochemical changes occurring during theactivation process that allow the immune cells to be stimulated toactivity in response to low doses of stimulants.

Immune cell activity that may be measured include, but is not limitedto, (1) cell proliferation by measuring the cell or DNA replication; (2)enhanced cytokine production, including specific measurements forcytokines, such as IFN-γ, GM-CSF, or TNF-α; (3) cell mediated targetkilling or lysis; (4) cell differentiation; (5) immunoglobulinproduction; (6) phenotypic changes; (7) production of chemotacticfactors or chemotaxis, meaning the ability to respond to a chemotactinwith chemotaxis; (8) immunosuppression, by inhibition of the activity ofsome other immune cell type; and, (9) apoptosis, which refers tofragmentation of activated immune cells under certain circumstances, asan indication of abnormal activation.

As used herein, the terms “cancer,” “neoplasm,” and “tumor,” are usedinterchangeably and in either the singular or plural form, refer tocells that have undergone a malignant transformation that makes thempathological to the host organism. Primary cancer cells (that is, cellsobtained from near the site of malignant transformation) can be readilydistinguished from non-cancerous cells by well-established techniques,particularly histological examination. The definition of a cancer cell,as used herein, includes not only a primary cancer cell, but any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by procedures such as CAT scan, MR imaging,X-ray, ultrasound or palpation, and/or which is detectable because ofthe expression of one or more cancer-specific antigens in a sampleobtainable from a patient.

Several different ways, to assess maturity and cell differentiation, areavailable. For example, one such method is by measuring cell phenotypes.The phenotypes of immune cells and any phenotypic changes can beevaluated by flow cytometry after immunofluorescent staining usingmonoclonal antibodies that will bind membrane proteins characteristic ofvarious immune cell types.

A second means of assessing cell differentiation is by measuring cellfunction. This may be done biochemically, by measuring the expression ofenzymes, mRNA's, genes, proteins, or other metabolites within the cell,or secreted from the cell. Bioassays may also be used to measurefunctional cell differentiation or measure specific antibody productiondirected at a patient's tumor, tumor cell lines or cells from freshtumors.

As used herein, “fresh tumors” refer to tumors removed from a host bysurgical or other means.

As mentioned above, T cells from patients are, preferably activated exvivo either by soluble anti-CD3 antibody, or are co-activated by usinganti-CD3 and anti-CD28 monoclonal antibodies, either by soluble orimmobilized on a solid support. A preferred solid support are plastics,or any surface upon which antibodies can be immobilized, or beads, suchas, for example, Dynal beads. Once activated, T cells are armed with abispecific antibody.

Bispecific antibodies are able to bind to the T cell receptor complex ofthe T cell with one binding arm and to tumor-associated antigens on thetumor cell with the second binding arm. Thereby, they activate T cellswhich kill tumor cells by releasing cytokines. Moreover, there is thepossibility that T cells recognize tumor-specific antigens via theirreceptor during activation by bispecific antibodies and that, along-lasting immunization is initiated. Of particular importance in thisregard is the intact Fc portion of the bispecific antibody whichmediates the binding to accessory cells such asmonocytes/macrophages/dendritic cells and causes these cells to becomecytotoxic themselves and/or at the same time to transduce importantco-stimulatory signals to the T cell.

Bispecific antibodies (BiAbs) have been used for targeting drugs,pro-drug activation, and immune recruitment strategies.⁹⁶ They candirectly mediate cytotoxicity to the tumor by specifically targeting theT cells to a tumor. BiAbs have been modified to bear enzymes for theconversions of circulating inactive pro-drug to active drug at the tumorsite. Infused BiAbs have been used to recruit and redirect immuneeffector cells to target tumor cells in vivo, and/or arm effector Tcells after ex vivo expansion for immunotherapy.

Intact bispecific antibodies are composed of two antibody semi-molecules(one H and one L immunoglobulin chain each) each representing aspecificity, and in addition have, like normal antibodies, a Fc portionperforming the well-known effector functions. A particularly preferredmethod for the preparation of bispecific antibodies, of the presentinvention, is by chemical heteroconjugation, as described in theExamples which follow. It should be understood that other methods ofpreparation are also useful if they lead to the intact bispecificantibodies according to the above definition required according to theinvention.

The immunoglobulins can have two pairs of light chain/heavy chaincomplexes, typically at least one chain comprising mouse complementaritydetermining regions functionally joined to human framework regionsegments. For example, mouse complementarity determining regions, withor without additional naturally-associated mouse amino acid residues,can be used to produce human-like antibodies capable of binding to Her2⁺ type tumors.

The intact bispecific antibodies used in the present invention carry afunctional Fc part Contrary to bispecific F(ab)2 fragments which do notinclude a functional Fc part the intact bispecific antibodies of thepresent invention are able to bind not only to T cells but alsoaccessory cells which are also known as Fc-receptor positive cells (e.g.monocytes, macrophages, dendritic cells). The binding of the cells playsan essential role in providing an efficient, direct tumor destructionwhich is 10-1000 times higher compared to the efficiency of the methodused by Kaneko, T. et al. Blood (Mar. 1, 1993) 81 (5): 1333-1341; andKaneko, T. et al. Leukemia and Lymphoma (1994) 14: 219-229; Kaneko, T.et al, Bone Marrow Transplantation (1994) 14: 213-217. The intactbispecific antibodies of the present invention enable an optimalco-stimulation of T cells. Particularly preferred surface antigens foroptimal co-stimulation are CD3 and/or CD28 and particular secretedcytokines (like IL-2, IL-6, IL-12, TNF-alpha).

Arming activated T cells using the intact bispecific antibodies of thepresent invention, efficiently directs destruction of tumor cells by Tcells. The presence of accessory cells, such as dendritic cells, maycause such cells to be, also, bound by the bispecific antibody of thepresent invention during the arming process. Dendritic cells from acancer patient can thus, be stimulated to uptake, process and presentparts of the tumor.

The immune mechanisms leading to destruction of target tumor cells areat least partially understood. A population of cytolytic T cells havebeen identified which carry the CD8+ antigenic determinant on theirsurfaces. These cells require CD4+ helper lymphocytes for activation,which is a complex event mediated by antigen processing and presentationin association with the major histocompatibility complexes. Antigenprocessing assures that only cells targeted to the tumor antigens willbe activated.

Monoclonal antibodies directed to various markers on subpopulations of Tlymphocytes have been used to activate immune effector cells. OKT3antibody administered by injection, for example, meets with CD3, and cancause a whole array of immune effects including release of IL-2,TNF-alpha, GM-CSF, MIP1α, RANTES, and/or IL-6, tissue damage, and eitheractivation or suppression of T cell activity. More recently, OKT3specificity has been combined with a antitumor specificity in abispecific antibody. Link, et al., Blood, 81: 3343 (1993) showed that abispecific antibody having one arm of OKT3 and the other arm directed toa B-cell malignant antigen was able to induce cytotoxicity of targettumor cells. Interestingly, the T-cell activation was without regard tothe natural specificity of the T cell, and required the presence of thetumor cells. Thus, in the simultaneous binding of tumor cell andeffector cell by the same antibody, the T cells are effectivelyrecruited from the general T cell population, and retargeted to destroythe tumor cells.

It has also been shown by Weiner, Int. J. Cancer, Supplement 7, 63(1992) that the action of the bispecific antibody is enhanced byco-administration of IL-2, so that combinational therapy resulted inmanagement of a 100 to 1000 times greater tumor load than with theanti-tumor monoclonal antibody alone. Alternatively, the co-stimulusobserved in the use of the bispecific antibody may be provided throughbinding of the Fc domain of the antibody to the Fc monocyte receptor,which in turn provide the co-stimulus, possibly through binding of theB7 family of membrane proteins to CD28. Preactivation ex vivo ofcytotoxic T cells with co-administration of bispecific F(ab') has alsobeen reported (Mezzanzanice, et al., Cancer Res., 51:5716 (1991)).

However, the present invention differs from the prior art in that the Tcells are activated and then armed with bispecific antibodies whosespecificity is directed against a tumor antigen, like for example Her2+tumors, prior to re-infusion into the patient.

In a preferred embodiment, the cellular composition of the reinfusion iscomprised of naïve T and B cells and accessory cells such as dendriticcells. Dendritic cells are powerful antigen presenting cells. Withoutwishing to he bound by theory, a composition of cells which includedendritic cells, for example between about 5×105 to about 2×106dendritic cells would provide a powerful antigen presenting cell so thatthe dose of armed T cells and/or naïve immunocompetent T cells could bereduced depending on the patient's prognosis. Thus, these stimulatetumor specific T cell responses from the pool of naïve T cells.

Monoclonal antibodies used in the production of bispecific antibodiesare available from commercial sources, for example anti-CD3 monoclonalantibody (OK-T3) is available from OrthoBiotech. Monoclonal antibodiesspecific for tumor antigens such as Her2+, (Herceptin®) can be purchasedfrom Genentech, S.F., Calif.

Monoclonal antibodies may also be produced in the laboratory. Thus, inselecting an antibody specific for a common antigenic determinantdisplayed on the cell surface of cancers of a defined cell type, amixture of cells is prepared, the mixture comprising cells fromindividual cell lines derived from a plurality of cancer cells ofdefined tissue type. This mixture of whole cells is then injected into alaboratory animal such as a mouse, according to a conventionalimmunization protocol to immunize the animal with the heterologous humantumor cells. Reactive B cells are then harvested from the animal,preferably the disrupted spleens, and fused with myeloma cells to formhybridomas. By maintaining the cell density below a critical level inwhich a statistical distribution function predicts one or two hybridomasper well, the likelihood of obtaining isolated single hybridomas isimproved.

After cloning and outgrowth, supernatant medium containing the secretedmonoclonal antibodies is removed. The screenings can then be carriedout, first, by contacting the mixture of cancer cells of the definedtissue, and a mixture of cancer cells of a different tissue type, withthe monoclonal antibody under conditions conducive to binding of theantibody to cells displaying the target antigenic determinant. Afluorescent dye that recognizes the antibody is then added and the cellsare then evaluated in a flow cytometer to determine which cells havedetectable dye and which do not. The cell types are distinguished by alog scale of emission light intensity. Thus, the cells are ranked into afirst class having labeled antibody bound to the surfaces thereof andinto a second class having no labeled antibody bound, thereby showing abimodal distribution of cells in flow cytometry.

The second screen involves further screening tests on the cells showinga bimodal distribution in which individual cells of prostate cancer andother cells of cancer origin are labeled with the monoclonal antibody.Thus, each cancer cell type is individually tested with the labeledantibody to identify antibody with binding specificity for the cancercells derived from the tissue of interest. Those antibodies whichdemonstrate unambiguous reactivity with, for example, prostatecancer-derived cells and no reactivity with nonprostate-derived cellsare further tested. The cancer tissue types for which this method isintended in its therapeutic application include all those derived orarising from body organs unessential for viability such as ovary,breast, certain endocrine glands (thyroid), testicle, as well asprostate.

The third screening test is performed upon the monoclonal antibodiespassing both the first and second screen, and involves determining thebinding specificity of the labeled antibody for tissue sections derivedfrom a plurality of cancers of defined cell type, together with controlsof normal tissue sections from nonhomologous tissue.

The term “derived” as it applies herein means the cells were obtained bysubculture of tumors isolated from patients. It also applies to celllines established from non-solid tumors of the lymphatic system. Thetechniques for routine subculture of tumor cells are well known in theart, and include the use of growth factors, nutrients, support matrices;and hormones, as required for the particular tissue type. The techniquesfor immunization of experimental animals and subsequent cell fusion ofsplenic B cells to produce hybridomas, and their subsequent culture areconventional. The basic protocol utilized in the practice of the presentinvention is set forth in detail in Current Protocols in Immunology,vol. 1, J. E. Coligan, et al., eds., John Wiley & Sons: 1991, herebyincorporated by reference.

It is important in applying these protocols to the isolation ofhybridomas according to the present invention, that a proper dilution offused cells occurs, so that a substantial number of wells in the 96 welltrays contain about 1-2 clones, and preferably, not more. At dilutionssufficiently great to attain this objective, some 6 to 12 percent ofwells will contain 0 clones.

Screening by flow cytometry has several key advantages. First, it isimportant that a stable cell surface antigen be identified. By selectingonly those antibodies that bind whole cells, the likelihood of choosinga stable surface component antigen is enhanced. The term “stable” meansin the context of antibody/cellular interactions, that the targetmolecule is preferably a constitutive cell membrane glycoproteinintegral to the structure and integrity of the membrane, and not atransient resident of the cell which is shed, displaced, orantigenically modified during the cell cycle.

A second advantage to screening by flow cytometry, is that the bimodalprofile indicates that some cells bind the fluorophore labeled antibodyand not others, which is a threshold indication of specificity. If onlya single fluorescent peak is observed, this means that some antigencommon to both the tumor cells and the non-prostate cells has beenidentified by the antibody. Two peaks mean that either one or moresubsets of tumor cells have a unique antigen, one or more subsets oftumor cells but not all share an antigen with the non-tumor cells, orthat the tumor cells have an antigen not shared by normal cells. Anotheradvantage of this method of pre-selection is that the techniques oflabeling cells and preparing them for flow cytometry are well known, andmay be carried out routinely.

Bispecific antibodies have been utilized in a variety of therapeuticapplications. U.S. Pat. No. 5,601,819 (Wong) discloses the use of acombinational CD3, and CD28 or interleukin 2 receptor bispecificantibody to selectively cause proliferation and destruction of specificT cell subsets. Belani, et al. showed that bispecific IgG functions in aB cell lymphoma model to retarget the specificity of T cells in lowdose, and to cause nonspecific T cell activation with systemic cytokineproduction at higher doses. It was found that bsF(ab')2 was also capableof retargeting T-cell mediated lysis by activated T cells. Thus, in manyapplications portions of antibodies, such as enzyme digested fragments,will mediate the effects otherwise observed for the intact antibody.These fragments necessarily contain the complementarity determiningregions (CDRs) of the variable light and heavy chain antibody domains,and may be integrated with other protein fragments to form a bispecificantigen binding protein construct. This construct will minimally containthe CDRs including the interspersed constant framework beta sheetportions. These regions are easily identified following routine cloningand sequencing procedures, as disclosed in U.S. Pat. Nos. 5,530,101 and5,585,089, hereby incorporated by reference. Cloning may be facilitatedby PCR primers complementary to conserved sequences flanking thefunctional variable regions.

Useful bispecific antibodies combining a CDR specific for an effectorcell and the CDR for a tissue specific antigen may also be humanized,either by replacing the light and heavy chain constant regions of themurine antibody with their human counterparts, or by grafting the CDRsonto a human antibody. Methods for carrying out these procedures arecontained in U.S. Pat. Nos. 5,530,101 and 5,585,089. The immuneconstruct of the present invention may also be bispecific single chainantibodies, which are typically recombinant polypeptides consisting of avariable light chain portion covalently attached through a linkermolecule to the corresponding variable heavy chain portion, as disclosedin U.S. Pat. Nos. 5,455,030, 5,260,203, and 4,496,778, herebyincorporated by reference. A more complex construct for a single chainbispecific antibody also containing an Fc portion is provided in detailin U.S. Pat. No. 5,637,481. The principal advantage of constructs ofthis type is that only one species of antibody is produced, rather thanthree separate antibody types in the fused cell hybrid-hybridoma, whichrequire further purification.

Other methods can be utilized in producing bispecific antibodies. Aparticular preferred method in the present invention is by chemicalheteroconjugation of two monoclonal antibodies. Monoclonal antibodies,in addition to the described methods of production may be purchased froma commercial source. Chemical heteroconjugates can be created by thechemical linking of either intact antibodies or antibody fragments ofdifferent specificities. The preferred method for chemicalheteroconjugation is described in the example section which follows andthe method is schematically shown in FIG. 1.

Bispecific antibodies may also be created by disulfide exchange, whichinvolves enzymatic cleavage and reassociation of the antibody fragments.Glennie et al., “Preparation and Performance of Bispecific F(ab')₂Antibody Containing Thioether Linked Fab' Fragments”, J. Immunol. 139:2367-2375 (1987). Another method is the creation of F(ab')₂ connectedvia a shortened Fc to the leucine zipper region of the transcriptionfactors Fos and Jun. Kostelny et al., “Formation of a BispecificMonoclonal Antibody by the Use of Leucine Zippers”, J. Immunol. 148:1547-53 (1992).

Bispecific antibodies arc also produced by hybrid-hybridomas.Hybrid-hybridomas are created by fusing two hybridoma cell linestogether so that the resulting hybrid-hybridoma contains two productivelight chain alleles. Hybrid-hybridomas secrete individual bispecific IgGmolecules which are monovalent for each of the two distinct antigensrecognized by antibodies produced by the parent hybridomas. However,hybrid-hybridomas produce both bispecific antibodies and monospecificantibodies for each of the two antigens recognized by the parenthybridomas. Further, light chain/heavy chain fidelity does not alwaysoccur. In all, there are 10 possible heavy and light chain combinationsthat could be produced by the hybrid-hybridoma cell line. Only one ofthese is the desired bispecific antibody. Some degree of purification ofthe bispecific component is therefore necessary prior to the use of suchbispecific antibodies. A preferred method of purification is protein Aimmunoaffinity chromatography followed by HPLC purification.

In order that the invention may be more completely understood, severaldefinitions are set forth. As used herein, the term “immunoglobulin”refers to a protein consisting of one or more polypeptides substantiallyencoded by immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 Kd or 214 amino acids) are encoded by a variableregion gene at the NH2-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), arcsimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

One form of immunoglobulin constitutes the basic structural unit of anantibody. This form is a tetramer and consists of two identical pairs ofimmunoglobulin chains, each pair having one light and one heavy chain.In each pair, the light and heavy chain variable regions are togetherresponsible for binding to an antigen, and the constant regions areresponsible for the antibody effector functions. In addition toantibodies, immunoglobulins may exist in a variety of other formsincluding, for example, Fv, Fab, and F(ab')₂, as well as bifunctionalhybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105(1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), andHunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporatedherein by reference).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled CDR's. The extent of the framework region and CDR's have beenprecisely defined (see, “Sequences of Proteins of ImmunologicalInterest,” E. Kabat et al., U.S. Department of Health and HumanServices, (1983); which is incorporated herein by reference). Thesequences of the framework regions of different light or heavy chainsare relatively conserved within a species. As used herein, a “humanframework region” is a framework region that is substantially identical(about 85% or more, usually 90-95% or more) to the framework region of anaturally occurring human immunoglobulin. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDR's. TheCDR's are primarily responsible for binding to an epitope of an antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to human constant segments, such asgamma 1 and gamma 3. A typical therapeutic chimeric antibody is thus ahybrid protein composed of the variable or antigen-binding domain from amouse antibody and the constant or effector domain from a humanantibody, although other mammalian species may be used.

As used herein, the term “humanized” immunoglobulin refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor.”Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody as defined above, e.g., because the entirevariable region of a chimeric antibody is non-human. One says that thedonor antibody has been “humanized”, by the process of “humanization”,because the resultant humanized antibody is expected to bind to the sameantigen as the donor antibody that provides the CDR's.

It is understood that the humanized antibodies may have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. By conservativesubstitutions are intended combinations such as gly, ala; val, ile, leu;asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

Humanized immunoglobulins, including humanized antibodies, have beenconstructed by means of genetic engineering. Most humanizedimmunoglobulins that have been previously described have comprised aframework that is identical to the framework of a particular humanimmunoglobulin chain, the acceptor, and three CDR's from a non-humandonor immunoglobulin chain.

A principle is that as acceptor, a framework is used from a particularhuman immunoglobulin that is unusually homologous to the donorimmunoglobulin to be humanized, or use a consensus framework from manyhuman antibodies. For example, comparison of the sequence of a mouseheavy (or light) chain variable region against human heavy (or light)variable regions in a data bank (for example, the National BiomedicalResearch Foundation Protein Identification Resource) shows that theextent of homology to different human regions varies greatly, typicallyfrom about 40% to about 60-70%. By choosing as the acceptorimmunoglobulin one of the human heavy (respectively light) chainvariable regions that is most homologous to the heavy (respectivelylight) chain variable region of the donor immunoglobulin, fewer aminoacids will be changed in going from the donor immunoglobulin to thehumanized immunoglobulin. Hence, and again without intending to be boundby theory, it is believed that there is a smaller chance of changing anamino acid near the CDR's that distorts their conformation. Moreover,the precise overall shape of a humanized antibody comprising thehumanized immunoglobulin chain may more closely resemble the shape ofthe donor antibody, also reducing the chance of distorting the CDR's.

Typically, one of the 3-5 most homologous heavy chain variable regionsequences in a representative collection of at least about 10 to 20distinct human heavy chains will be chosen as acceptor to provide theheavy chain framework, and similarly for the light chain. Preferably,one of the 1-3 most homologous variable regions will be used. Theselected acceptor immunoglobulin chain will most preferably have atleast about 65% homology in the framework region to the donorimmunoglobulin.

In many cases, it may be considered preferable to use light and heavychains from the same human antibody as acceptor sequences, to be surethe humanized light and heavy chains will make favorable contacts witheach other. Regardless of how the acceptor immunoglobulin is chosen,higher affinity may be achieved by selecting a small number of aminoacids in the framework of the humanized immunoglobulin chain to be thesame as the amino acids at those positions in the donor rather than inthe acceptor.

Humanized antibodies generally have at: least three potential advantagesover mouse or in some cases chimeric antibodies for use in humantherapy:

1) Because the effector portion is human, it may interact better withthe other parts of the human immune system (e.g., destroy the targetcells more efficiently by complement-dependent cytotoxicity (CDC) orantibody-dependent cellular cytotoxicity (ADCC)).2) The human immune system should not recognize the framework orconstant region of the humanized antibody as foreign, and therefore theantibody response against such an antibody should be less than against atotally foreign mouse antibody or a partially foreign chimeric antibody.

Antibodies can also be genetically engineered. Particularly preferredare humanized immunoglobulins that are produced by expressingrecombinant DNA segments encoding the heavy and light chain CDR's from adonor immunoglobulin capable of binding to a desired antigen, such asthe human T cell CD3 complex, attached to DNA segments encoding acceptorhuman framework regions.

The DNA segments typically further include an expression control DNAsequence operably linked to the humanized immunoglobulin codingsequences, including naturally-associated or heterologous promoterregions. Preferably, the expression control sequences will be eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells, but control sequences for prokaryotic hosts mayalso be used. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the humanized light chains, heavy chains,light/heavy chain dimers or intact antibodies, binding fragments orother immunoglobulin forms may follow (see, S. Beychok, Cells ofImmunoglobulin Synthesis, Academic Press, New York, (1979), which isincorporated herein by reference).

Human constant region DNA sequences can be isolated in accordance withwell known procedures from a variety of human cells, but preferablyimmortalized B-cells (see, Kabat op. cit. and WP87/02671). The CDR's forproducing preferred immunoglobulins of the present invention will besimilarly derived from monoclonal antibodies capable of binding to thepredetermined antigen, such as the human T cell receptor CD3 complex,and produced by well known methods in any convenient mammalian sourceincluding, mice, rats, rabbits, or other vertebrates, capable ofproducing antibodies. Suitable source cells for the constant region andframework DNA sequences, and host cells for immunoglobulin expressionand secretion, can be obtained from a number of sources, such as theAmerican Type Culture Collection (“Catalogue of Cell Lines andHybridomas,” sixth edition (1988) Rockville, Md., U.S.A., which isincorporated herein by reference).

Other “substantially homologous” modified immunoglobulins to the nativesequences can be readily designed and manufactured utilizing variousrecombinant DNA techniques well known to those skilled in the art. Forexample, the framework regions can vary at the primary structure levelby several amino acid substitutions, terminal and intermediate additionsand deletions, and the like. Moreover, a variety of different humanframework regions may be used singly or in combination as a basis forthe humanized immunoglobulins of the present invention. In general,modifications of the genes may be readily accomplished by a variety ofwell-known techniques, such as site-directed mutagenesis (see, Gillmanand Smith, Gene, 8, 81-97 (1979) and S. Roberts et al., Nature, 328,731-734 (1987), both of which are incorporated herein by reference).

Substantially homologous immunoglobulin sequences are those whichexhibit at least about 85% homology, usually at least about 90%, andpreferably at least about 95% homology with a reference immunoglobulinprotein.

Alternatively, polypeptide fragments comprising only a portion of theprimary antibody structure may be produced, which fragments possess oneor more immunoglobulin activities (e.g., complement fixation activity).These polypeptide fragments may be produced by proteolytic cleavage ofintact antibodies by methods well known in the art, or by inserting stopcodons at the desired locations in vectors known to those skilled in theart, using site-directed mutagenesis.

As stated previously, the DNA sequences can be expressed in hosts afterthe sequences have been operably linked to (i.e., positioned to ensurethe functioning of) an expression control sequence. These expressionvectors are typically replicable in the host organisms either asepisomes or as an integral part of the host chromosomal DNA. Commonly,expression vectors contain selection markers, e.g., tetracycline orneomycin resistance, to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which isincorporated herein by reference).

E. coli is one prokaryotic host useful particularly for cloning the DNAsequences of the present invention. Other microbial hosts suitable foruse include bacilli, such as Bacillus subtilus, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(see, Winnacker, “From Genes to Clones,” VCH Publishers, New York, N.Y.(1987), which is incorporated herein by reference). Eukaryotic cells areactually preferred, because a number of suitable host cell lines capableof secreting intact immunoglobulins have been developed in the art, andinclude the CHO cell lines, various COS cell lines, HeLa cells,preferably myeloma cell lines, etc, and transformed B-cells orhybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer (Queen et al., Immunol. Rev., 89, 49-68 (1986), which isincorporated herein by reference), and necessary processing informationsites, such as ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences. Preferred expressioncontrol sequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, cytomegalovirus, Bovine Papilloma Virus, and the like.

The vectors containing the DNA segments of interest (e.g., the heavy andlight chain encoding sequences and expression control sequences) can betransferred into the host cell by well-known methods, which varydepending on the type of cellular host. For example, calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment or electroporation may be used for other cellularhosts. (See, generally, Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, (1982), which is incorporated hereinby reference.)

Once expressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like (see, generally, R Scopes, “ProteinPurification”, Springer-Verlag, N.Y. (1982)). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred, for pharmaceuticaluses. Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically (includingextracorporeally) or in developing and performing assay procedures,immunofluorescent staining, and the like. (See, generally, ImmunologicalMethods, Vols. I and II, Lefkovits and Pernis, eds., Academic Press, NewYork, N.Y. (1979 and 1981)).

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward et al.(1989) Nature 341:544-546, each of which is hereby incorporated hereinby reference. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced, seeCabilly, U.S. Pat. No. 4,816,567. These patents are incorporated hereinby reference.

The antibodies of this invention can also be used for affinitychromatography in isolating a protein or antigenic target. Columns canbe prepared where the antibodies are linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysatemay be passed through the column, the column washed, followed byincreasing concentrations of a mild denaturant, whereby the purifiedantigen will be released.

The antibodies may also be used to screen expression libraries forparticular expression products, for example anti-CD3 or any othermolecule that can activate a T cell and be used in arming the ATC.Usually the antibodies used in such a procedure will be labeled with amoiety allowing easy detection of presence of antigen by antibodybinding.

Antibodies raised against, for example, a CD3 complex will also heuseful to raise anti-idiotypic antibodies.

The preparation and characterization of the preferred phage-displayedrandom peptide libraries have been described elsewhere. See, forexample, Kay, B. K. et al. in Gene (1992) 128:59-65, for a descriptionof the preparation of the phage-displayed random peptide library knownas TSAR-9, more below. In particular, by cloning degenerateoligonucleotides of fixed length into bacteriophage vectors, recombinantlibraries of random peptides can be generated which are expressed at theamino-terminus of the pIII protein on the surface of M13 viralparticles. (There are 3-5 copies of the pIII-fusion on the surface ofeach particle.) Consequently, these libraries can be screened byisolating viral particles that bind to targets. The isolates can begrown up overnight, and the displayed peptide sequence responsible forbinding can be deduced by DNA sequencing.

These libraries have approximately >10⁸ different recombinants, andnucleotide sequencing of the inserts suggests that the expressedpeptides are indeed random in amino acid sequence. These libraries arereferred to herein as TSAR libraries, where TSAR stands for TotallySynthetic Affinity Reagents. The preparation of the TSAR libraries aredescribed further in U.S. Pat. No. 6,432,920.

A preferred pharmaceutical composition of the present inventioncomprises the use of the monoclonal antibodies, which are eithercommercially available or produced by the methods described above. Themonoclonal antibodies are preferably chemically heteroconjugated toproduce the bispecific antibody of interest. These bispecific antibodiesare then used to “arm” the activated T cells.

In one embodiment, the bispecific antibody remains bound to the T cellafter the armed cell has killed a tumor target, thereby allowing thearmed T cell to kill multiple target tumors, i.e. the bispecificantibody does not become unbound from the armed T cell, once the armed Tcell has contacted and killed a tumor target. The armed T cell, once ithas killed one tumor target can engage another tumor target for whichthe armed T cell is specific, kill the second tumor target and proceedto engage another tumor target etc. Preferably, the armed T cellperforms multiple rounds of target cell killing. This is discussed indetail in the Examples which follow.

As used herein, a “target cell” is any cell comprising antigens that thearmed T cell binds to. The target cell is not limited to tumor antigensbut can include for example, viral antigens, infectious disease organismantigens and the like.

As used herein, “arming” is the binding of the bispecific antibodyportion specific for the T cell antigen of interest, that is the T cellreceptor complex antigens such as CD3 and/or CD3 and CD28. The secondportion of the bispecific antibody is the antibody which is specific forthe tumor antigen of choice, thereby targeting the activated T cell tothe specific tumor antigen.

While various procedures involving the use of antibodies have beenapplied in the treatment of tumors, few if any successful attempts usingcytotoxic T-cells have been recorded. Theoretically, cytotoxic T-cellswould be the preferable means of treating tumors. However, no procedureshave been available to specifically activate cytotoxic T-cells. Incontrast to antibodies, the T-cell receptors on the surface of CD8 cellscannot recognize foreign antigens directly. Antigen must first bepresented to the T cell receptor.

The present invention provides a detailed description of activation of Tcells as described in the examples which follow.

The presentation of antigen to CD8 T-cells is accomplished by majorhistocompatibility complex (MHC) molecules of the Class I type. Themajor histocompatibility complex (MHC) refers to a large genetic locusencoding an extensive family of glycoproteins which play an importantrole in the immune response. The MHC genes, which are also referred toas the HLA (human leukocyte antigen) complex, are located on chromosome6 in humans. The molecules encoded by MHC genes are present on cellsurfaces and are largely responsible for recognition of tissuetransplants as “non-self”. Thus, membrane-bound MHC molecules areintimately involved in recognition of antigens by T-cells.

MHC products are grouped into three major classes, referred to as I, II,and III. T-cells that serve mainly as helper cells express CD4 andprimarily interact with Class II molecules, whereas CD8-expressingcells, which mostly represent cytotoxic effector cells, interact withClass I molecules.

Class I molecules are membrane glycoproteins with the ability to bindpeptides derived primarily from intracellular degradation of endogenousproteins. Complexes of MHC molecules with peptides derived from viral,bacterial and other foreign proteins comprise the ligand that triggersthe antigen responsiveness of T-cells. In contrast, complexes of MHCmolecules with peptides derived from normal cellular products play arole in “teaching” the T-cells to tolerate self-peptides, in the thymus.Class I molecules do not present entire, intact antigens; rather, theypresent peptide fragments thereof, “loaded” onto their “peptide bindinggroove”.

As used herein, “allogeneic” is used to refer to immune cells derivedfrom non-self major histocompatibility complex donors. HLAhaplotypes/allotypes vary from individual to individual and it is oftenhelpful to determine the individual's HLA type. The HLA type may bedetermined via standard typing procedures and the peripheral bloodlymphocytes (PBLs) purified by Ficoll gradients.

As will be recognized by those in the art, the term “host compatible” or“autologous” cells means cells that are of the same or similar haplotypeas that of the subject or “host” to which the cells are administered.

As used herein, “partially-mismatched HLA”, refers to HLA types that arebetween about 20 to 90% compatible to the host's HLA type.

For many years, immunologists have hoped to raise specific cytotoxiccells targeting viruses, retroviruses and cancer cells. While targetingagainst viral diseases in general may be accomplished in vivo byvaccination with live or attenuated vaccines, no similar success hasbeen achieved with retroviruses or with cancer cells. Moreover, thevaccine approach has not had the desired efficacy in immunosuppressedpatients. At least one researcher has taken the rather non-specificapproach of “boosting” existing CD8 cells by incubating them in vitrowith IL-2, a growth factor for T-cells. However, this protocol (known asLAK cell therapy) will only allow the expansion of those CD8 cells whichare already activated. As the immune system is always active for onereason or another, most of the IL-2 stimulated cells will be irrelevantfor the purpose of combating the disease. In fact, it has not beendocumented that this type of therapy activates any cells with thedesired specificity. Thus, the benefits of LAK cell therapy arecontroversial at best, and the side effects are typically so severe thatmany studies have been discontinued.

The presentation of Class I MIIC molecules bound to peptide alone hasgenerally been ineffective in activating CD8 cells. In nature, the CD8cells are activated by antigen-presenting cells, such as, for example,dendritic cells, which present not only a peptide-bound Class I MHCmolecule, but also a costimulatory molecule. Such costimulatorymolecules include B7 which is now recognized to be two subgroupsdesignated as B7.1 and B7.2. It has also been found that cell adhesionmolecules such as integrins assist in this process.

Dendritic cells are antigen-presenting cells that arc found in alltissues and organs, including the blood. Specifically, dendritic cellspresent antigens for T lymphocytes, i.e., they process and presentantigens, and stimulate responses from naive and memory T cells. Inaddition to their role in antigen presentation, dendritic cells directlycommunicate with non-lymph tissue and survey non-lymph for an injurysignal (e.g., ischemia, infection, or inflammation) or tumor growth.Once signaled, dendritic cells initiate the immune response by releasingIL-1 which triggers lymphocytes and monocytes.

When the CD8 T-cell interacts with an antigen-presenting cell, such as adendritic cells, having the peptide bound by a Class I MHC andcostimulatory molecule, the CD8 T-cell is activated to proliferate andbecomes an effector T-cell. See, generally, Janeway and Travers,Immunobiology, published by Current Biology Limited, London (1994),incorporated by reference.

Accordingly, what is needed and which the present invention provides, isa means to activate T-cells so that they proliferate and becomecytotoxic. The present invention is also useful as the activation isdone in vitro and the activated cytotoxic T-cells reintroduced into thepatient. Activation is achieved by the crosslinking of the T cellreceptor complex (anti-CD3 and anti-CD28 antibodies) which increase theeffectiveness of the activation.

The present invention also provides for the involvement of antigenpresenting cells as the reinfused composition of cells is comprised ofarmed activated T cells, naïve T and B cells and dendritic cells.Besides the capacity of being powerful antigen presenting cells, thedendritic cells can also bind to the Fc portion of the bispecificantibody, thereby forming a complex of armed T cells bound to the tumorand dendritic cells bound to the Fc portion of the bispecific antibody.Therefore, an extremely powerful, localized immune response istheoretically produced by the engagement of antigen presenting cellssuch as dendritic cells at the tumor site.

Chemokines and cytokines also play a powerful role in the development ofan immune response. The role of chemokines in leukocyte trafficking isreviewed by Baggiolini (1998) Nature 392:565-8, in which it is suggestedthat migration responses in the complicated trafficking of lymphocytesof different types and degrees of activation will be mediated bychemokines. The use of small molecules to block chemokines is reviewedby Baggiolini and Moser (1997) J. Exp. Med. 186:1189-1191.

The role of various specific chemokines in lymphocyte homing has beenpreviously described. For example, Campbell et al. (1998) Science,showed that SDF-1 (also called PBSF), 6-C-kine (also called Exodus-2),and MIP-3beta (also called ELC or Exodus-3) induced adhesion of mostcirculating lymphocytes, including most CD4+ T cells; and MIP-3alpha(also called LARC or Exodus-1) triggered adhesion of memory, but notnaive, CD4+ T cells. Tangemann et al. (1998) J. Immunol. 161:6330-7disclose the role of secondary lymphoid-tissue chemokine (SLC), a highendothelial venule (HEV)-associated chemokine, with the homing oflymphocytes to secondary lymphoid organs. Campbell et al. (1998) J. CellBiol 141(4):1053-9 describe the receptor for SLC as CCR7, and that itsligand, SLC, can trigger rapid integrin-dependent arrest of lymphocytesrolling under physiological shear.

Preferably, the ATC or COACT induce memory T cells from naïve orpluripotent stem cells. Immature and mature T cells are readilyidentifiable by markers and can be detected by flow cytometric analysis.A review of the biology of memory T cells may be found in Dutton et al.(1998) Ann. Rev Immunol 16:201-23. Memory cells express a differentpattern of cell surface markers, and they respond in several ways thatare functionally different from those of naive cells. Human memory cellsare CD45RA⁻, CD45RO⁺. In contrast to naive cells, memory cells secrete afull range of T cell cytokines.

Mature B cells can be measured in immunoassays, for example, by cellsurface antigens including CD19 and CD20 with monoclonal antibodieslabeled with fluorochromes or enzymes may be used to these antigens. Bcells that have differentiated into plasma cells can be enumerated bystaining for intracellular immunoglobulins by direct immunofluorescencein fixed smears of cultured cells.

Several different ways, to assess maturity and cell differentiation, areavailable. For example, one such method is by measuring cell phenotypes.The phenotypes of immune cells and any phenotypic changes can beevaluated by flow cytometry after immunofluorescent staining usingmonoclonal antibodies that will bind membrane proteins characteristic ofvarious immune cell types.

A second means of assessing cell differentiation is by measuring cellfunction. This may be done biochemically, by measuring the expression ofenzymes, mRNA's, genes, proteins, or other metabolites within the cell,or secreted from the cell. Bioassays may also be used to measurefunctional cell differentiation or measure specific antibody productiondirected at a patient's tumor, tumor cell lines or cells from freshtumors.

As used herein, “fresh tumors” refer to tumors removed from a host bysurgical or other means.

Immune cells express a variety of cell surface molecules which can bedetected with either monoclonal antibodies or polyclonal antisera.Immune cells that have undergone differentiation or activation can alsobe enumerated by staining for the presence of characteristic cellsurface proteins by direct immunofluorescence in fixed smears ofcultured cells.

In vitro T cell cytotoxic assays are well known to those skilled in theart. In general, cytotoxicity is measured in a 5 hr ⁵¹Sodium chromate(⁵¹Cr) release assay. In particular, a 20 hr ⁵¹Cr-release assay ispreferred. Tumor cells, also referred to herein as “target cells” areplated in flat-bottomed microtiter plates and incubated at 37° C.overnight. The targets are washed and labeled the next day with ⁵¹Cr at37° C. ⁵¹Cr is taken up by the target cells, either by endocytosis orpinocytosis, and is retained in the cytoplasm. The wells containingtumor cells are washed, and then armed or unarmed ATC, referred to as“effector cells” are plated at different E:T ratios and incubatedovernight at 37° C. Cytolysis is a measure of the ⁵¹Cr released from thetarget cells into the supernatant due to destruction of the target cellsby the effector cells. The microtiter plates are centrifuged at 1000 rpmfor 10 minutes and an aliquot of about 50 μl to about 100 μl is removedand the level of radioactivity is measured the next day by a gammacounter and the percent specific lysis calculated.

Percent specific lysis is measured by using the formula:

(⁵¹Cr released from the target cells)−(spontaneous ⁵¹Cr released fromthe target cells)/(maximum ⁵¹Cr released from the targetcells)−(spontaneous ⁵¹Cr released from the target cells)×100

The spontaneous ⁵¹Cr released from the target cells is measured withtumor cells to which no effector cells have been added. Maximum ⁵¹Crreleased from the target cells is obtained by adding, for example, 1MHCl and represents the total amount of ⁵¹Cr present in the cytoplasm ofthe target cell.

All of the cytotoxicity assays shown in this application are conductedwithout the addition of IL-2 for 18 to 20 hours. The redirectedcytotoxicity mediated by armed ATC or COACTS occurs in presence of serumand complement and in the absence of IL-2. Therefore, the infused armedT cells are likely to kill tumor for at least 18 to 20 hours in theabsence of IL-2.

Serum and complement do not affect cytotoxicity mediated by armed ATC.Armed ATC were not lysed in the presence of fresh PBMC and rabbitcomplement or high concentrations of fresh human serum. This resultsuggests that armed ATC are not lysed in vivo by complement fixation andlysis via Fc-receptor mediated antibody dependent cellular cytotoxicity.

Other cytotoxicity assays such as the labeling of target cells withtritiated thymidine (³H-TdR) may also be used. ³H-TdR is taken up bytarget cells into the nucleus of the cell. Release of ³H-TdR is ameasure of cell death by DNA fragmentation. The assay is conducted asabove except the incubation period is at least about 48 hours and 50 μlto about 100 μl of the supernatant is measured by a beta-counter in thepresence of at least about 1 ml of scintillation fluid. Calculation ofpercent specific lysis is performed using the above formula

The following definitions are used throughout the application:

The term “fluorescent component” or “fluorescent label” or “labeled”refers to a component capable of absorbing light and then re-emitting atleast some fraction of that energy as light over time. The term includesdiscrete compounds, molecules, naturally fluorescent proteins andmacro-molecular complexes or mixtures of fluorescent and non-fluorescentcompounds or molecules. The term “fluorescent component” or “fluorescentlabel” also includes components that exhibit long lived fluorescencedecay such as lanthanide ions and lanthanide complexes with organicligand sensitizes, that absorb light and then re-emit the energy overmilliseconds. Other labels include different fluorochromes andfluorescent proteins such as green fluorescent protein. Fluorochromeswhich may find use in a multicolor analysis include phycobiliproteins,e.g., phycoerythrin and allophycocyanins; fluorescein and Texas red.

Activated T Cells (ATC) are a heterogeneous population of humanlymphocytes predominantly T lymphocytes of CD8 phenotype that have beentriggered to proliferate after stimulation with OKT3 and grown in lowdoses of IL-2. ATC has been safe given in combination with subcutaneousor continuous infusion of IL-2 at low doses of 300,000 IU/m²/day andGM-CSF at doses as high as 250 μg/m²/day or as low as 125 μg/m²/twiceweekly. ATC may also be administered with other immune augmentingcytokines or chemokines, such as for example, IL-12.

Preferably, reinfused ATC or COACT are administered to a patient twice aweek for about four weeks. The skilled practitioner will be able todetermine the correct times and doses depending on the age, sex, bodyweight and condition of the patient.

Murmonab OKT3: OKT3 (OrthoClone OKT3) has been extensively characterizedin both preclinical and clinical testing. OKT3 is a murine IgG2a MAbdirected at human CD3 and is commercially available from OrthoBiotech,Raritan, N.J. It is purchased in 5 mg vials containing 5 ml ofbacteriostatic water. It is used to produce ATC, coat Dynal beads forthe production of COACTS, and produce chemical heteroconjugate with 9184or Herceptin®.

IL-2 Proleukin: Proleukin (recombinant IL-2) is purchased from Chiron,Emeryville, Calif. It is approved for the treatment of renal cellcarcinoma. The clinical grade drug is used to expand T cells in culture.It is currently being used in this laboratory to expand ATC and COACTS.

COACTS are a heterogeneous population of human T cells, roughly 50% ofthe CD4+ phenotype, that have been triggered to proliferate anddifferentiate with coimmobilized OKT3 and 9.3 Mabs. COACTS are grownwith or without low doses of IL-2 up to 14 days. Only the COACTS areinfused into patients. IL-2, OKT3, 9.3, and the beads are not present inthe final product. OKT3 is coimmobilized with 9.3 (anti-CD28 monoclonalantibody) to costimulate T cells.

Murm 9.3 is a mouse Mab of the IgG2a isotype directed at the CD28receptor on human T cells. The 9.3 Mab was produced under clinical gradecondition by Abbott-Biotech. The antibody is coimmobilized with OKT3 onDynal beads to activate T cells during culture. The antibody is notinfused into patients. The monoclonal antibody is removed when the beadsare removed.

The BiAb: OKT3×9184 or OKT3×Herc [either designated as Her2Bi] are usedto arm ATC so that the non-MHC restricted cytotoxicity exhibited by ATCcan be redirected by the bispecific antibody to lyse targets expressinga specific target antigen. ATC armed with Hcr2bi are designated ATCarmed with Her2Bi.

Clinical grade 9184 Mab: Clinical material, GMP 9184 (anti-Her-2/neu),is supplied in vials containing 1 mg by Nexell Corporation, Irving,Calif. 9184 has been extensively characterized by Nexell and has beenused for purging of stem cell products in the European market.

Anti-Her2/neu monoclonal antibody (9184, Nexell Corporation). Anti-Her2(9184 monoclonal provided as a study drug by Nexell Corporation is amurine IgG1 monoclonal antibody directed at Her2/neu. The bindingcharacteristics and its ability to mediate redirected cellularcytotoxicity towards MCF-7, SK-BR-3 and PC-3 have been well documentedin our preclinical studies.

Her2/neu is a tumor associated antigen (TAA) on prostate cancers.Her2/neu (Her2) belongs to the epidermal growth factor receptor familyof tyrosine kinases. The Her2/neu oncogene encodes an 185 kDatransmembrane receptor with significant sequence homology to class Ireceptor tyrosine kinase family.¹⁴⁵ Her2 is over-expressed in breast,ovarian, lung, gastric, oral,¹⁴⁶ and prostate cancers.¹⁴²⁻¹⁴⁴ Theover-expression of Her2 makes it an ideal target. The expression ofHer2/neu on prostate cancer, however, is controversial. There arereports that suggest that expression of Her2/neu on prostate cancers islow or absent,^(142;147;148) or high.¹⁴⁴

Herceptin®: Herceptin® (Genentech, SF, Calif.) has been extensivelycharacterized in preclinical and clinical trials. The Mab iscommercially available for in vivo use for the treatment of stage IVbreast cancer in combination with Taxol.¹⁶¹

OKT3: Clinical grade OKT3 obtained commercially will be heteroconjugatedto 9184 or Herceptin® to produce OKT3×9184 or OKT3×Herc, respectively.

Armed T Cells (The “Drug”):

Armed ATC are ATC grown for 6-14 days and armed with Her2Bi (OKT3×9184or OKT3×Herc).

Armed COACTS are COACTS grown for 6-14 days and armed with Her2Bi(OKT3×9184 or OKT3×Herc).

For certain therapeutic applications, a DNA expression vector encoding adesired cytokine, such as, for example, IL-12, chemokine, or any otherimmune-augmenting molecule of the invention can be introduced intoimmune cells of the present invention such as, for example, T cells.

The term “vector” as used herein (including “expression vector”) meansany nucleic acid sequence of interest capable of being incorporated intoa host cell resulting in the expression of a nucleic acid of interest.Vectors can include, e.g., linear nucleic acid sequences, plasmids,cosmids, phagemids, and extrachromosomal DNA. Specifically, the vectorcan be a recombinant DNA. Also used herein, the term “expression”, or“gene expression”, is meant to refer to the production of the proteinproduct of the nucleic acid sequence of interest, includingtranscription of the DNA and translation of the RNA transcript.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/04701, which has a targeting moiety (e.g. a ligand toa cellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. Viral vectors can be chosen tointroduce the cytokine or chemokine to cells of choice. Such vectorsinclude pox vectors such as orthopox or avipox vectors, herpesvirusvectors such as herpes simplex I virus (HSV) vector (Geller, A. I et al,J. Neurochem., 64:487 (1995); Lim, F., et al., in DNA Cloning: MammalianSystems, D. Glover, Ed. (Oxford Univ. Press, Oxford, England) (1995);Geller, A. I. et al., Proc. Natl. Acad. Sci. USA 87:1149 (1990))Adenovirus vectors (LeGal LaSalle et al., Science, 259:988 (1993);Davidson, et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol.69:2004 (1995)) and Adeno-associated virus vectors (Kaplitt, M. G. etal., Nat. Genet. 8:148 (1994)).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only short term expression of the nucleic acid.Adenovirus vectors, adeno-associated virus vectors and herpes simplexvirus vectors are preferred for introducing the nucleic acid into neuralcells. The adenovirus vector results in a shorter term expression (about2 months) than adeno-associated virus (about 4 months), which in turn isshorter than HSV vectors. The vectors can be introduced by standardtechniques, e.g. infection, transfection, transduction ortransformation. Examples of modes of gene transfer include for example,naked DNA calcium phosphate precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection andviral vectors.

In a preferred embodiment, armed ATC are used in the treatment ofhormone refractory prostate cancer (HRPC) and other patients with Her2+tumors. However, the present invention allows for treatment of all typesof tumors as a bispecific antibody can be generated with specificity fora tumor present in an individual patient. The present invention isadvantageous in that it allows for use of autologous T cells andmonoclonal antibody generation for a specific tumor present in anindividual patient. Thus, treatment can be tailored for each individualpatient and allows for changes in treatment if the tumor antigenchanges, by generation of new monoclonal antibodies against the newtumor antigen. The new tumor specific antibody can then beheteroconjugated to the T cell activating antibody to form a newbispecific antibody, specific for the new tumor antigen and then used toarm the autologous ATC or COACT and reinfused into the patient.

In particular, the invention provides for determination of an individualpatient's maximum tolerated dose (MTD) of armed ATC, involving patientswith HRPC as well as determining the effectiveness of the armingstrategy on an individual patient basis.

Preferably, the protocol for treatment of a patient requires theisolation of peripheral blood mononuclear cells (PBMC). PBMC's areisolated on a Ficoll-Hypaque gradient. PBMC's from each patient areactivated as described in the examples which follow and thencryopreserved. Twice per week, ATC or COACTS are thawed, washed, armedwith Her2bi, and infused into the patient. Preferably, there are fourdose levels in the dose escalation schedule of armed ATC. The doselevels are at least about 2, 3, 5, and 8 billion armed ATC/infusion(total dose range of about 20-80 billion armed ATC/infusion). Patientsare started on a very low dose (determination of doses are described inthe examples which follow). If cell-based toxicity occurs, the doselevel is decreased.

In accordance with the invention, suitable dose ranges of armedATC/infusion are between about 2.5 billion armed ATC to about 40 billionarmed ATC. Infusions of the present invention are administered at leastabout twice per week for at least about four weeks. A preferred armedATC/infusion dose is at least about 2.5 billion, more preferably about10 billion, most preferably about 40 billion armed ATC/infusion.

Toxicities are discussed infra. If the patient shows no signs ofcell-based toxicity then the dose level is advanced to the next doselevel. Preferably, at least about ten infusions are given to eachpatient. About two doses are infused per week for two weeks and aboutone dose per week for about 6 weeks. The patients also receivesubcutaneous IL-2, or other immune augmenting cytokines, such as IL-12,of at least about 300,000 IU/m²/day, preferably on a daily basisbeginning about 3 days before the first infusion and until about 1 weekafter the last infusion of armed ATC or COACTS.

Preferred dose ranges of IL-2 are at least about 50,000 IU/m²/day, morepreferably about 150,000 IU/m²/day, most preferably about 300,000IU/m²/day.

Preferred dose ranges of IL-12, or other immune augmenting cytokines orchemokines, are at least about 50,000 IU/m²/day, more preferably about150,000 IU/m²/day, most preferably about 300,000 IU/m²/day. These dosesmay vary depending on the age, sex, condition, size, weight etc, of apatient, as determined by a practitioner well-skilled in the art.

GM-CSF is administered at a dose of about 125 μg/m², administered atleast about twice per week beginning about 3 days before the firstinfusion and until about 1 week after the last infusion of armed ATC orCOACTS. A preferred dose range of GM-CSF is at least about 50 μg/m²,more preferably about 100 μg/m², and most preferably about 125 μg/m².Tumor and immune evaluations are performed at about 0, 3, 6, 9 and 12months. If clinical response occurs, the patient is retreated at thenext dose level. ¹¹¹Indium labeling of armed and unarmed ATC or COACTScan be used to determine trafficking of armed and unarmed ATC or COACTS.The trafficking procedure is usually conducted after the MTD has beendetermined.

The above procedure is an example of how the treatment protocol is usedfor patients with hormone refractory prostate cancer, but can bemodified according to the type of tumor to be treated. The amount ofarmed ATC or COACTS administered to the patient will also vary dependingon the condition of the patient and should be determined viaconsideration of all appropriate factors by the practitioner.Preferably, however, about 1×10⁶ to about 1×10¹², more preferably about1×10⁸ to about 1×10¹¹, and even more preferably, about 1×10⁹ to about1×10¹⁰ armed ATC or COACTS cells are utilized for adult humans. Theseamounts will vary depending on the age, weight, size, condition, sex ofthe patient, the type of tumor to be treated, the route ofadministration, whether the treatment is regional or systemic, and otherfactors. Those skilled in the art should be readily able to deriveappropriate dosages and schedules of administration to suit the specificcircumstance and needs of the patient.

Methods of re-introducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example,administration of activated CD8 cells via intravenous infusion isappropriate.

As used herein, determination of whether immunotherapy with armed ATC orCOACTS can induce clinical remissions (CR) is measured by a 50%reduction of the appropriate tumor antigen. For example, clinicalremission in a patient with hormone refractory prostate cancer isdefined by a 50% reduction in the serum PSA level, which is determinedby conventional ELISAs.

As used herein, determination of whether immunotherapy with armed ATC orCOACTS can improve overall survival (OS), is defined as the length oftime from day of entry into immunotherapy treatment until death.

As used herein, determination of whether immunotherapy with armed ATCCOACTS can improve progression free survival (PFS), is defined as thelength of time from day of entry into immunotherapy treatment untilprogression of disease.

In the present invention, there have been no dose-limiting toxicities ofIL-2 given at doses of 300,000 IU/m²/day (continuous infusion orsubcutaneously) and GM-CSF at 125 μg/kg/day given subcutaneously whengiven in combination with multiple doses of ATC after PBSCT for stage111b/IV breast cancer. Based on the clinical toxicities seen with theuse of Herceptin alone or in combination with chemotherapy¹⁵⁹, ATC armedwith ng amounts of BiAb do not produce dose-limiting cell-basedtoxicities. One can expect the side effects that are seen with theinfusion of monoclonal antibodies such as fever, chills, low bloodpressure, wheezing, and shortness of breath that can be treated bydecreasing the dose level and time between infusions or byadministration of antihistamines, for example, Benedryl™.

In a preferred embodiment, for the activation of ATC or COACTS, anti-Tcell receptor monoclonal antibodies are preferably used. Preferredantibodies used to activate T cells, OKT3 for the production of ATC andOKT3/9.3 coated on a solid support, such as Dynal beads, to produceCOACTS. Both ATC and COACTS are thoroughly washed during the harvestprocedure to remove any medium components.

The production of ATC preferably involves a single pulse of antibody,followed by washing the cell product free of any remaining antibodyprior to cell infusion, as discussed in the examples which follow.

The preferred production of COACTS only involves the Dynal beads bearingthe coimmobilized antibodies OKT3 and 9.3, which are removed on aboutday 4 of in vitro culture. The minimum release criteria for the COACTSis preferably, at least about less than 25 beads/3×10⁶, more preferablyat least about less than 50 beads/3×10⁶ cells, and most preferably atleast about less than 100 beads/3×10⁶.

The activation method described herein, differs from the prior art inthat the present invention directly cross-links the T cell receptor witha monoclonal antibody and expands the activated T cells in low dose IL-2as described in the examples which follow. For example, autolymphocytetherapy (ALT) involves infusions of autologous PBMC produced by culturescontaining extracts of autologous tumor and conditioned media (CM)derived from PBMC stimulated with OKT3.⁵⁰ Preclinical studies inC57BL/6J mice using tumor extracts from Lewis lung carcinoma and B16melanoma showed that splenocytes can be stimulated to expand CD44⁺memory CTL and respond to tumor challenge.^(51;52) Subsequent studiesusing ALT generated cytotoxic effector cells could be obtained from PBMCgrown in CM produced by OKT3-stimulated PBMC instead of autologoustumor. This procedure differs from the present invention in that ALT isdistinguished from anti-CD3 activated T cells (ATC) based on differencesin the activation method. ALT is indirectly activated via supernatantsfrom OKT3-stimulated PBMC. In contrast, ATC are prepared by directlycross linking the T cell receptor (TCR) with OKT3 and expanding the Tcells in low dose IL-2.

Anti-CD3 activated T cells (ATC): Cross linking of the T cell receptor(TCR) with anti-CD3 monoclonal antibody (MAb) leads to T cellproliferation, cytokine synthesis, and immune responses.⁵⁴⁻⁵⁷ ATC areproduced by OKT3 stimulation of PBMC in the presence of low doses ofIL-2 (at least about 5-100 IU/ml). ATC have a variety of clinicalapplications. ATC have activated NK and NK-like cytotoxic properties,produce tumoricidal cytokines, and can serve as vehicles that candeliver targeting antibodies or gene products. Preclinical studies showthat ATC can be expanded from PBMC or bone marrow from normals andpatients with malignancy and mediate non-MHC restrictedcytotoxicity.⁶²⁻⁷² In vitro studies showed that human ATC exhibitednon-MHC restricted cytotoxicity to Daudi cells (ANK sensitive targets),K562 cells (NK sensitive targets), leukemic blasts,^(73;74)neuroblastomas,⁶³ and autologous plasma cells in multiple myeloma.⁷⁵ Inaddition, ATC produce immunologically active or tumoricidal cytokinessuch as IFNγ, TNFα, or GM-CSF.

Anti-CD3/Anti-CD28 coactivated T Cells (COACTS): Cross linking of theTCR with anti-CD3 triggers a signaling cascade resulting in T cellproliferation, cytokine synthesis, and immune responses.⁵⁴⁻⁵⁷ However,optimal activation and proliferation requires costimulation of CD28receptors on T cells with anti-CD28 or B7 molecules (CD80 andCD86).⁷⁹⁻⁸³ These interactions enhance proliferation and stabilizationof mRNAs for IL-2, IFNγ, TNFα, and granulocyte-macrophage colonystimulating factor (GM-CSF).⁸⁴ Costimulation of the CD28 receptor alsoleads to enhanced production of beta chemokines RANTES, MIP1-α andMIP1-β⁸⁵ The enhanced secretion of chemokines at the tumor site mayaugment recruitment of effector cells.

In one preferred embodiment, ATC or COACTS can be transduced withvectors coding for chemokines to deliver locally, high concentrations ofcytokines which recruit other effector cells, trigger T cellproliferation and enhance a localized immune response. Methods fortransduction are well known in the art.

A “vector” is a composition which can transduce, transfect, transform orinfect a cell, thereby causing the cell to express nucleic acids and/orproteins other than those native to the cell, or in a manner not nativeto the cell. A cell is “transduced” by a nucleic acid when the nucleicacid is translocated into the cell from the extracellular environment.Any method of transferring a nucleic acid into the cell may be used; theterm, unless otherwise indicated, does not imply any particular methodof delivering a nucleic acid into a cell. A cell is “transformed” by anucleic acid when the nucleic acid is transduced into the cell andstably replicated. A vector includes a nucleic acid (ordinarily RNA orDNA) to be expressed by the cell. A vector optionally includes materialsto aid in achieving entry of the nucleic acid into the cell, such as aviral particle, liposome, protein coating or the like. A “celltransduction vector” is a vector which encodes a nucleic acid capable ofstable replication and expression in a cell once the nucleic acid istransduced into the cell.

Anti-CD3/anti-CD28 coactivated T cells (COACTS) exhibit in vitroanti-tumor activity directed at a variety of tumor cell lines.⁸⁶ COACTSproduce Th₁-type cytokine profiles^(79;87) and may survive longer invivo due to induction of the cell survival gene Bcl-x₁, which confersresistance to apoptosis.^(88;89)

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLES Materials and Methods Components Used for ATC or COACTS.

Orthoclone OKT3 (muromonab-CD3). OKT3 is purchased from Ortho-Biotech(Raritan, N.J.). OKT3 is supplied as a sterile solution in packages of 5ampoules (NDC 59676-101-01) containing 5 mg of muromonab-CD3. Proleukin®(Aldesleukin, IL-2). Proleukin® (recombinant IL-2) is purchased from.Chiron (Emeryville, Calif.). ATC are expanded in the presence of lowdose IL-2 (100 IU/ml) in RPMI 1640 (BioWhittaker) supplemented with 2-5%human serum (BioWhittaker). RPMI 1640 (BioWhittaker) is supplementedwith 2 mM L-glutamine (BioWhittaker), and 50 μg/ml gentimicin(BioWhittaker).

“Armed ATC” or “Armed COACTS” are the best descriptive names for thearmed and activated T cells. The chemically heteroconjugatedanti-CD3×anti-Her2 (either OKT3×9184 or OKT3×Herceptine) is referred toas Her2bi. Therefore, ATC armed with anti-CD3 x anti-Her2/neu BiAb aredesignated Her2bi armed ATC or COACTS. OKT3×Herceptin® has beenabbreviated to OKT3×Herc.

Preparation of Anti-CD3×Anti-Her2 Bispecific Antibody.

Equimolar concentrations of OKT3 and anti-Her2 (9184 or Herceptin) areconjugated. OKT3 is reacted with Traut's reagent at room temperature(RT) for 1 hr and 9184, Herceptin, or control irrelevant antibodies arereacted with sulphosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate at RT. Both Mabs arepurified on 10 DG columns (Biorad) in PBS to remove unbound crosslinker. The cross-linked Mabs are mixed at equimolar ratios andconjugated at 4° C. overnight. At these concentrations, dimer formationis optimal and multimer formation is minimized. The reactants, products,and purified fractions of the heteroconjugation reaction are visualizedby SDS non-reducing PAGE and Coomassie blue staining. The final productsOKT3×9184 or OKT3×Herc are cleared for final use only after 7 days ofbacteria and fungal cultures, PCR for mycoplasma (ATCC, Catalog#90-1001K), and assay for endotoxin (BioWhittaker, Catalog #50-6470) areall negative.

Each bispecific antibody heteroconjugate lot (previous testing negativefor bacteria, fungi, Mycoplasma, and endotoxin) is tested by adose-titration arming of normal cryopreserved ATC or COACTS againstPC-3, MCF-7, and/or SK-BR-3 target cells prior to release. Multiplevials of pretested normal donors have been expanded for 14 days,cryopreserved in aliquots for lot testing as well as providing normalcontrols in the evaluation of the armed patient ATC. Each lot mustexhibit a dose-titration effect with increased specific cytotoxicity atthe same E/T ratio when as the arming dose of the Her2Bi is increased.The arming dose range of each lot is determined by such a titration. Thelot is rejected if a % specific cytotoxicity of atleast about 50% ofPC-3, MCF-7 or SK-BR-3 at an E/T of 25:1 can not be attained after a 20hr cytotoxicity assay with an arming dose of 50 ng/million ATC orCOACTS.

Preparation, Culture, Arming and Cryopreservation of ATC or COACTS.

Peripheral blood mononuclear cells (PBMC) from normal subjects andcancer patients are isolated by Ficoll-Hypaque (Lymphoprep from NycomedPharma, Oslo, Norway). PBMC are activated on plates coated with 2 μg/mlimmobilized OKT3 or 20 ng/ml of soluble OKT3 (Ortho Biotech, Inc.,Raritan, N.J.).^(67;68) COACTS are produced from PBMC or a leukopheresisproduct by co-stimulating the PBMC with beads co-coated with OKT3 and9.3 Mabs for 4 days. After 4 days of culture, the beads are removed andthe cultures continued for an additional 10 days. Unless otherwiseindicated, ATC or COACTS are grown for 14 days in RPMI 1640 medium(BioWhittaker, Walkersville, Md.) supplemented with antibiotics,L-glutamine, 100 IU/ml of IL-2 (Chiron Corp., Emeryville, Calif.), and10% fetal calf serum FCS (Hyclone, Logan, Utah) or human serum(BioWhittaker) as indicated. Cells are counted and maintained at 10⁶/ml.Viability is determined by trypan blue exclusion. T cells are grown fromnormal subjects or cancer patients. The T cells are washed, counted, andincubated (“armed”) with BiAb at the indicated doses (10⁶ T cells for 1hr at 4° C. in 125 μl of media). The T cells are washed twice incomplete medium prior to testing.

Activation, Culture, Cryopreservation, Thawing, and Washing of ATC.

In brief, lymphocytes are obtained by leukopheresis, cultured at adensity of 1-3×10⁶ cells/ml in RPMI 1640 medium supplemented with 2 mMof L-glutamine, 100 IU/ml of IL-2, 10-20 ng/ml of OKT3-, and 2% pooledhuman serum-. Cells are cultured for a maximum of 14 days. The ATC arecounted, split, and/or fed every 3-4 days with complete medium based oncell concentration No additional OKT3 is added. After culture, ATC areharvested and washed using the Fenwal Cell harvester, and cryopreservedin 10% DMSO and 20% protein (albumin or autologous plasma) usingcontrolled rate freezing and storage in liquid nitrogen. No exogenousIL-2, OKT3, or other culture reagents (e.g. medium components) arepresent in the final cryopreserved product.

Activation, Culture, Bead Removal, Cryopreservation, Thawing, andWashing of COACTS.

In brief, lymphocytes are obtained by leukopheresis, cultured at adensity of 1-3×10⁶ cells/ml in Ex vivo 15 medium supplemented with 2 mMof L-glutamine, 100 IU/ml of IL-2, -and 2% pooled human serum-. PBMCfrom the leukopheresis produce are cocultured with Dynal beads withcoimmobilized GMP grade OKT3 and 9.3 in order to coactivate the T cells.The beads are removed using the MagSep device after 4 days of cultureand the COACTS are put back into culture. The COACTS are counted, split,and/or fed every 3-4 days with complete medium containing 100 IU/ml ofIL-2 final. After culture, COACTS are harvested and washed using theFenwal Cell harvester, and cryopreserved in 10% DMSO and 20% protein(albumin or autologous plasma) using controlled rate freezing andstorage in liquid nitrogen. No exogenous IL-2, OKT3, culture reagents(e.g. beads or medium components) are present in the final cryopreservedproduct.

Initiation, Splitting, and Harvest of ATC Cultures.

PBMC at a concentration of 1×10⁶ mononuclear cells/ml are activated with20 ng/ml of soluble OKT3 in RPMI 1640 supplemented with L-glutamine,gentimicin, 100 IU/ml of IL-2, and 2% human serum in Stericel MultipleContainer Sets. The activated T cells are counted split, and fed basedon their expansion rate. After 6-14 days of culture, the ATC areharvested. If the amount of ATC to be harvested is 1 to 10 liters,procedures well-known in the art will be used for small volume T cellharvest & Cryopreservation. For volumes exceeding 10 liters, the ATC areharvested using a Fenwal Cell Harvesting System. Cryopreservation ofunarmed or armed ATC is conducted using commercial human serum.

Anti-CD3 (OKT3) and anti-CD28 (9.3) monoclonal antibodies immobilized onbeads are used to cross link cellular receptors. OKT3 is purchased fromOrtho-Biotech (Raritan, N.J.). 9.3 antibody, lot #3-309-900411 wasproduced for Dr. Carl June by Abbott Biotech. OKT3 and 9.3 are linked toparamagnetic, polystyrene Dynabeads via tosyl chemistry in a 1:1stoichiometry as per the manufacturer's protocol. The magnetic beads andthe monoclonal antibodies linked to the beads are all produced under GMPconditions for clinical use.

COACTS are expanded in RPMI 1640 or X VIVO 15 supplemented with 2-5%human serum.

Initiation of Cultures Using OKT3/9.3 Coated Beads in 3 L Bags.

The procedure for seeding PBMC and stimulating PBMC with soluble OKT3(20 ng/ml) in the presence of 100 IU/ml of IL-2 to produce ATC ismodified to incorporate the introduction of OKT3/9.3 coated. The PBMCare counted, beads are added at a ratio of 3:1 (beads/cells), and themixed in seeded into 3 L gas-permeable bags supplied by NexellTherapeutics Inc (Irving, Calif.).

After the removal of beads on day 4 of culture using the MaxSep, theCOACTS are placed back into culture in the conditioned medium. TheCOACTS are harvested, washed, and concentrated using a Fenwal CellHarvester (Baxter).

Infusion Product Free of Carryover Cytokines.

The infused cell product does not contain exogenous cytokines. Data showthat the IL-2 used in culture is undetectable by ELISA after 1 wash ofATC in 50 ml tube. Before washing of the cultured ATC, 3.5 IU/ml of IL-2was detected. In a second experiment, duplicate 50 ml tubes spiked with1200, 600, and 300 IU of IL-2/ml had no detectable IL-2 after 1 wash.The ELISA is sensitive to 50 pg/ml (<1 IU/ml). Since both ATC and COACTSare washed equally, COACTS do have any biologically significantcytokines after the harvest/wash.

Components to Produce OKT3×9184 or OKT3×Herceptin®.

This application uses two BiAbs. Both BiAbs target Her2/neu use thechemical heteroconjugation process to produce the combination ofanti-CD3×anti-Her2/neu.

OKT3×9184 consists of clinical grade Orthoclone OKT3 (muromonab, IgG2amurine Mab directed at CD3 is purchased from Ortho-Biotech, Raritan,N.J.) chemically heteroconjugated to clinical grade 9184 (IgG1 murineMab directed at Her2/neu is a gift from Nexell, Corporation, Irving,Calif.).

OKT3×Herceptin (OKT3×Herc), consists of clinical grade OKT3 (IgG2amurine monoclonal antibody directed at CD3) chemically heteroconjugatedto clinical grade Herceptin® (trastzumab, a humanized IgG1 Mab directedat Her2/neu is purchased from Genentech, San Francisco, Calif.).

Traut's Buffer: Traut's Buffer (pH 8) consists of Triethanolamine (SigmaUltra #T9534), 1.5 M NaCl (Sigma), 1 mM EDTA (Disodium Dihydrate)(Sigma)

Sulphosuceinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate:Sulpho-SMCC (SMCC buffer, Pierce #22322) is purchased from Pierce,Rockford, Ill.

Heteroconjugation Products: Equimolar quantities of OKT3 crosslinkedwith Traut's buffer and Sulpho-SMCC crosslinked 9184, Herceptin, orRituxan are incubated overnight at 4° C. A non-reducing SDS gel isperformed to identify the presence of monomer, dimer, or multimer. Thematerial is sterile filtered and quality control by sterility,endotoxin, and Mycoplasma testing using standard procedures and releasecriteria (Limulus Amebocyte Lysate, BioWhittaker Catalog #50-6470;Mycoplasma detection kit, ATCC, Catalog #90-1001K).

ATC Armed with OKT3×9184 or OKT3×Herc.

ATC are thawed after cryopreservation, armed with a pretitrated doseranging from 5-100 ng of unpurified chemically heteroconjugated Her2Biper million ATC, washed free of non-binding antibodies, and infused intothe patients.

COACTS Armed with OKT3×9184 or OKT3×Herc.

COACTS are thawed after cryopreservation, armed with a pretitrated doseranging from 5-100 ng of unpurified chemically heteroconjugated Her2Biper million, and washed free of non-binding antibodies. The armed COACTSare infused into the patients. There have been no toxicities or sideeffects attributed to antibody or bead carryover in patients whoreceived up to 60×10⁹ COACTS.

Specific Arming Dose for Each Patient.

The optimal arming dose is determined for each patient based ontitration of a frozen aliquot of the patient's ATC or COACTS cellproduct. The arming dose is adjusted to achieve a % specificcytotoxicity level at an E/T of 25:1 of at least 30% on PC-3, SK-BR-3,or MCF-7 cells. The arming dose for each patient is recorded on thearming worksheet for each patient. Table 1, below, is an illustrativeexample of a treatment schedule and is not meant to limit or construethe invention in any way.

Treatment Schema

The treatment includes subcutaneous injections of IL-2 beginning 3 daysbefore the first armed ATC or COACTS infusion at a dose of 3×10⁵IU/m²/day. IL-2 will be given until-7 days after the last dose of armedATC or COACTS.

The treatment includes subcutaneous injections of 125 μg/m² of GM-CSFtwice per week for 8 weeks beginning 3 days before the first armed ATCinfusions and ending 7 days after the last dose of armed ATC or COACTS.

ATC infusions in the presence of low dose IL-2 in refractory cancers(SLMC BRM 94-01, BRM 95-02) and the combination of PBSCT for metastaticbreast cancer (BRM 95-03) followed by ATC infusions, continuous infusionIL-2 (about 3×10⁵ IU/m²/day for about 65 days), and GM-CSF injections(between about day 5 and about day 21 after PBSCT) have not inducedlife-threatening autologous graft-versus-host disease or autoimmunesyndrome. The grade III skin rashes induced by the combination of ATC,IL-2, and GM-CSF may or may not have been autologous graft-versus-hostdisease or autoimmune syndromes (GVHD). Patients may develop an“autologous or syngeneic” GVHD skin rash due to dysregulated oroveractive T cells. Two of 23 patients who received PBSCT and ATCdeveloped skin rashes thought to be related to autologous GVHD but couldhave been related to chemotherapy agents such as Taxol. No treatment wasrequired and the skin rashes spontaneously resolved. The rashes were nottreated with steroids. No patient has suffered irreversible toxicitiesdue to the syndrome.

Arming of the Clinical Product.

From 3 to 10 billion (20% more than the targeted amount is processed toaccount for washing and processing losses) cryopreserved ATC or COACTSare thawed, washed, resuspended in plasmanate containing 2% albumin, andarmed in centrifuge tubes by incubating for 1 hr at 4° C. The Her2Bi iswashed twice in plasmanate containing 2% albumin, and transferred into atransfer bag for infusion into the patient. An aliquot is removed forviability, sterility and cytotoxicity testing.

Tumor Cell Lines and Monoclonal Antibodies.

The breast cancer lines MCF-7 cells and SK-BR-3 are purchased from ATCCRockville, Md. OKT3 is purchased from OrthoBiotech. The 9184 is ananti-Her2/neu, IgG1 provided by Nexell. Herceptin® is purchased fromGenentech, SF, Calif.), Rituxan (anti-CD20) is purchased from Genentech.T84.55 hybridoma (anti-carcinoembryonic antigen) is purchased from ATCC,Rockville, Md. IG3 (anti-prostate specific membrane antigen) is a giftfrom Alton Boynton of Northwest Biotherapeutics, Seattle, Wash.

Table 2 summarizes the binding and functional characteristics of the twobispecific antibodies, OKT3×9184 and OKT3×Herc, for rosetting, flowcytometry, and cytotoxicity.

TABLE 2 Cell Lines MCF-7 SK-BR-3 PC-3 Rosetting OKT3 × 9184 4+ 4+ 3+OKT3 × Herc 4+ 4+ 3+ Flow Cytometry OKT3 × 9184 1+ 4+ 2+ OKT3 × Herc 1+4+ 2+ Specific Cytotoxicity OKT3 × 9184 3+ 4+ 4+ OKT3 × Herc 4+ 4+ 4+Prostate Carcinoma, Breast Carcinoma, and Other Her2/neu+ Tumors.

The patients undergo leukopheresis for lymphocytes for generating ATC.At the designated times for infusions, ATC are thawed, washed, armedwith Her2bi, and infused. There arc four dose levels in the doseescalation schedule of armed ATC. Each dose level has 3 patients. Thedose levels are 2, 3, 5, and 8 billion/infusion (total dose of 20-80billion). If cell-based toxicity occurs, three additional patients willbe added to the same dose level before advancing to the next dose level.Ten infusions are given to each patient. Two doses are infused per weekfor two weeks and one dose per week for the subsequent 6 weeks. Thepatients receive subcutaneous IL-2 (300,000 IU/m²/day) daily beginning 3days before the first infusion and until 1 week after the last infusionof armed ATC. GM-CSF is given at a dose of 125 μg/m² twice per weekbeginning 3 days before the first infusion and until 1 week after thelast infusion of armed ATC. Tumor and immune evaluations are performedat 0, 3, 6, 9 and 12 month after the first iii armed ATC infusion.Indium labeling of armed and unarmed ATC is conducted in selectedpatients with measurable disease to determine if arming of ATC improvestrafficking to tumor sites.

Adverse events are scored using the NCI immunotherapy toxicity scoringsystem. A 50% decline in the PSA level is considered an objectiveresponse. PSA levels, Her2/neu receptor levels, IFNγ ELISPOTS by PBMCbefore and after infusions, IFNγ ELISPOTS responses to autologous tumor(if available), cytokine secretion, phenotyping of PBMC, T cellproliferation, and HAMA responses are evaluated at the designated timepoints.

Administration of low dose subcutaneous (SQ) IL-2 (2×10⁵ IU/m²/day) for90 days resulted in no grade III toxicities. None of the patientsexperienced pulmonary capillary leak syndrome, severe hypotension,oligouria, azotemia, or hyperbilirubinemia.¹⁶² The most frequenttoxicities included fatigue, fever, and nausea. None of the patients hadto stop their SQ IL-2 therapy due to side effects. Therefore, it isunlikely that major toxicities associated with the low dose SQ IL-2occur, although death due to high dose IL-2 is a known toxicity.

GM-CSF is a colony stimulating factor. Known Side Effects andToxicities: Patients receiving GM-CSF (Leukine-Sagra-mostim) haveexperienced fever 60-90 min after administration (duration 1-4 hrs);chills; nausea; vomiting; diarrhea; fatigue; weakness; headache;decreased appetite; thrombosis; rapid or irregular heartbeat or otherheart problems; feeling of faintness; facial flushing; pain in thebones, muscles, chest, abdomen, or joints; local reaction at the site ofinjection; rashes; and kidney and liver dysfunction. Eosinophilia orother blood component abnormalities may occur. There have beeninfrequent reports of fluid accumulation or worsening of preexistingfluid accumulation in the extremities, in the lungs, and around theheart which may result in breathing problems or heart failure. Rarely,patients have developed acute allergic reactions. There have also beenreports of low blood pressure, hypoxia, transient loss of consciousness,and difficulty in breathing after the first injection of Sargramostim.These signs may or may not recur with additional injections ofSargramostim. Patients with prior heart, lung, kidney, or liver problemsmay have worsening of their symptoms following administration ofSargramostim. There may be other side effects that could occur.

Infusions of Her2bi Armed ATC.

The ATC infusions are conducted on an outpatient basis in the BMT unit.All appropriate assurances for identification of product, patient,sterility, etc. are performed prior to reinfusion. Frozen ATC arethawed, washed, and then armed with a pretitrated dose of Her2bi. Thearmed ATC are washed 3 times and resuspended in infusion medium. Thetime for Her2bi armed ATC infusions varies from patient to patient, butthe dose of armed ATC (up to 8 billion) is given over 30 min. Allpatients are observed for a least 1 hr after an infusion. Vital Signs(T, P, R, and BP) are obtained before and every 15 min until the end ofthe observation period. All vitals are recorded on the patient'simmunotherapy toxicity scoring flowchart. If stable, the patient can bedischarged home.

Determination of Maximum Tolerated Dose (MTD)

The endpoints for the Phase I trial are defined as: 1) reaching themaximum tolerated dose of armed ATC or 2) reaching the technical limitof cell product expansion. Three patients arc entered at each doselevel. Each patient receives ten infusions per course of treatment. Thedose levels are 2, 3, 5, and 8 billion per infusion. The first threepatients will receive ten infusions of 2×10⁹ for a total dose of 2×10¹⁰armed ATC (1st dose level); the 2^(nd) three patients will receive teninfusions of 3×10⁹ for a total dose of 3×10¹⁰ armed ATC (2^(nd) doselevel); the 3^(rd) three patients will receive ten infusions of 5×10⁹for a total dose of 5×10¹⁰ armed ATC; and the 4^(th) three patients willreceive ten infusions of 8×10⁹ for a total dose of 8×10¹⁰ armed ATC. Thecell dose is increased until the MTD is reached. Two doses are infusedper week for the first two weeks and one dose per week for thesubsequent 6 weeks.

Dose Modification for Her2bi Armed ATC or COACTS.

If there is persistent grade 3 or more severe toxicity at any time, thetreatment will he held until toxicity improves to grade 0 or 1. If thetoxicities continue at the reduced dose of armed ATC or COACTS, the IL-2will be stopped and the armed ATC or COACTS will be continued at thereduced dose. If grade 3 or more severe toxicity again occurs, the armedATC or COACTS infusions will be stopped. Toxicity is assessed daily for2 days after each reinfusion and weekly between treatment courses forunresolved toxicities. Patients who develop non-hematological grade 4toxicity will be required to discontinue treatment.

Armed ATC or COACTS toxicities are toxicities that occur during thearmed ATC or COACTS infusions and up to 12 hrs after the infusions.Delayed clinical manifestations such as GVHD of the skin, liver, or gutor other autoimmune disease are considered as delayed toxicities of theinfusions for purposes of dose-escalation. However, systemic infectiondue to infusion of a contaminated cell product is not considered aproduct-related toxicity in the determination of the MTD.

Toxicity Grading—The NCI Common Toxicity Scale.

-   -   If Grade I-II toxicities occur, the patient may continue with        the infusion schedule.    -   If Grade III toxicity occurs, the “drug” will be held until the        toxicity decreases to Grade I or II, then the infusion will be        restarted. If Grade III or IV toxicity occurs after the restart,        the “drug” infusions will be stopped.    -   If Grade IV toxicity occurs, the patient is scored as having        Grade IV toxicity and the next infusion is reduced to the        previous dose. If the previous dose causes Grade IV toxicity,        then the “drug” will be stopped.    -   If Grade IV toxicity occurs in 1 of 3 patients at a specific        dose level, an additional 3 patients must be entered at that        cell-dose level for a total of 6 patients at that dose level. If        2 of 6 patients at a cell-dose level develop Grade IV toxicity,        this dose is defined as the MTD. The next 3 patients will be        given 66% (two-thirds) of the previous cell-dose level. For the        purposes of evaluation for dose-escalation, each patient at the        same dose level must have received at least 4 of 6 infusions.

Radioactively Indium Labeled Armed T Cells

The amount of radioactivity put onto the armed T cells is similar tothat used in bone scans.

After the MTD is determine, approximately 6 patients with measurabledisease will be entered into the trafficking study. Each patientreceives unarmed ¹¹¹Indium labeled ATC, scanned for tumor localizationevery 24 hrs for at least 72 hrs. After the disappearance of the labelon scanning, the same patient receives the same amount of ¹¹¹Indiumlabeled Her2Bi armed ATC to determine if there arc significantdifferences in amount of labeling at tumor sites. Both unarmed and theHer2Bi armed ATC are labeled with ¹¹¹Indium. After the MTD for Her2Biarmed ATC has been determined, selected patients will be studied withinfusions of ¹¹¹Indium labeled unarmed ATC. ATC are thawed, washed freeof freezing medium, armed or left unarmed with the optimal concentrationof Her2bi for 1 hr at 4° C., washed free of excess arming antibody, andthen labeled with ¹¹¹Indium using standard nuclear medicine labelingmethodology. Planar whole-body imaging is performed at 4, 24, 48, and 72hrs with a gamma camera (Picker Prism 2000, Picker International,Cleveland, Ohio) equipped with general purpose medium energycollimators. The patients are followed until the label can no longer bedetected. Imaging time is approximately 30 min with a target of 500 kcounts to be acquired at 4 hrs.

Stage IV Breast Cancer Treatments

Women with stage 1V breast cancer are entered into a phase I doseescalation trial comprised of increasing doses of the bispecificantibody OKT3×Herceptin (HER2Bi) armed, activated T cells (armed ATC).ATC are expanded for 14 days from a leukopheresis product, armed withHER2Bi, cryopreserved and infused in 8 divided doses. Escalating dosesof armed-ATC are given until the maximum tolerated dose is determined orthe maximum technically attainable dose has been reached. Three patientsat each dose level receive doses of 2.5, 5.0, 10, 20, and 40 billionarmed-ATC twice/week for 4 weeks. If there is a grade 3non-hematological toxicity in one patient at any dose level, then thecohort will be expanded to six patients at that dose level. The dose isescalated to the next level only if no more patients in the expandedcohort tolerate therapy without developing grade 3 non-hematologicaltoxicity. Low dose IL-2 (about 300,000 IU/m²/day) and GM-CSF (about 150μg/m² twice per week) are started one day prior to the first armed-ATCinfusion and end 7 days after the last dose of armed ATC.

After the maximum tolerated dose (MTD) is determined in the phase Isetting, a phase II clinical trial using the MTD is performed in 33patients with 2+ or 3+HER2/neu over-expressing breast cancer tumors.This allows for defining the toxicity profile; determine clinicalresponses; evaluation of overall and progression free survival;sequential evaluation of clinical and immune parameters. Selectedpatients with HER2/neu positive tumors in the phase I and II parts ofthe clinical trials are given ¹¹¹Indium-labeled unarmed and armed ATC todetermine the survival rate of the armed ATC and localization of armedATC to metastasized tumor sites.

ELISA Assay for Cytokines.

Armed or unarmed ATC or COACTS are cultured with or without PC-3, MCF-7,or SK-BR-3 as appropriate. ATC or COACTS are armed with OKT3×9184 orOKT3×Her and irrelevant controls (OKT3, 9184, Herc, and/or OKT3×Rituxan)at concentrations paralleling the concentrations used for the clinicalarming process. Armed ATC or COACTS are cocultured at E/T of 10:1 or 5:1for 24 hrs. The target and effector cells are adjusted to insure aplating concentration of 1×10⁶ of T cells. The supernatants are testedby ELISA and the amount of cytokine produced is expressed at pg/millionarmed ATC or COACTS/24 hrs.

ELISPOTS for Single Cell Secretion IFNγ.

Armed or unarmed ATC or COACTS are cultured with or without PC-3, MCF-7,or SK-BR-3 as appropriate. ATC or COACTS are armed with OKT3×9184 orOKT3×Her and irrelevant controls (OKT3, 9184, Herc, and/or OKT3×Rituxan)at concentrations paralleling the concentrations used for the clinicalarming process. Armed ATC or COACTS are cocultured at E/T of 10:1 or 5:1for 24 hrs. The target and effector cells are adjusted to insure aplating concentration of 1×10⁶ of T cells. The T cells in these culturesare harvested leaving the target cells on the flat-bottomed well. The Tcells are counted and plated onto ELISPOT wells (Millipore Corp.) andthe number of spots are enumerated using a dissecting microscope 24 hrslater. The number of IFNγ spots are expressed as IFNγ secretingcells/million armed ATC or COACTS.

¹¹¹Indium Oxine Labeling of Her2Bi Armed or Unarmed ATC.

A sample of ATC or Her2Bi armed ATC are labeled with ¹¹¹Indium Oxine(Amersham Healthcare, Arlington Heights, Ill.) using a modification ofstandard method.^(163;164) Approximately 2×10⁵ COACTS are washed andresuspended in buffered, glucose-containing medium and 700 μCi of¹¹¹Indium Oxine is mixed with the sample. The labeled cells are thenmixed with the remainder of the ATC dose and re-infused into thepatient.

Cytotoxicity Assay.

Cytotoxicity is measured in a 20 hr ⁵¹Cr-release assay. Tumor cells areplated in flat-bottomed microtiter plates and incubated at 37° C.overnight. The targets are washed and labeled the next day with ⁵¹Cr at37° C. The wells containing tumor cells are washed, and armed or unarmedATC are plated at different E:T ratios and incubated overnight at 37° C.The next day, the plates are counted in a gamma counter and the percentspecific lysis calculated.

All of the cytotoxicity assays shown in this application were conductedwithout the addition of IL-2 for 18 to 20 hours. The redirectedcytotoxicity mediated by armed ATC or COACTS occurs in presence of serumand complement and in the absence of IL-2. Therefore, the infused armedT cells are likely to kill tumor for at least 18 to 20 hours in theabsence of IL-2.

Serum and complement do not affect cytotoxicity mediated by armed ATC.Armed ATC were not lysed in the presence of fresh PBMC and rabbitcomplement or high concentrations of fresh human serum. This resultsuggests that armed ATC would not be lysed in vivo by complementfixation and lysis via Fc-receptor mediated antibody dependent cellularcytotoxicity.

Institutional Review Board.

The protocol entitled “Targeting of Her2/neu Prostate Carcinoma in MenWith Hormone Refractory Prostate Cancer Using Activated T Cells ArmedWith Anti-CD3×Anti-Her2/neu Bispecific Monoclonal Antibodies” and itsconsent form has been approved by the Roger Williams HospitalInstitutional Review Board. The IRB will monitor the study and IRBapproval will be obtained before changes of the protocol are initiated.All unanticipated problems involving risks to human subjects or otherproblems will be reported to the IRB.

The treatment plan will be according to the protocol(s), the therapyinvolved in the specific protocols and alternative forms of therapy willbe presented to patients by the Investigators or their designees. Therisk and hazards of the procedure will be fully explained to thepatient. The protocols will contain the specific details and the consentforms.

Summary of Preclinical Work

The preclinical studies show: The optimal time interval for arming ATCbegins around day 4 and persists up to day 14 after activation of ATC orCOACTS. The saturating arming dose of OKT3×9184 or OKT3×Herc needed foroptimal cytotoxicity is around 50 ng/10⁶ ATC or COACTS. Bispecificantibody mediated cytotoxicity is enriched in the dimer fraction basedon parallel studies done using OKT3×T84.66 (anti-CEA). Tumoricidalcytokine secretion by normal and patient ATC is induced by specificbinding to tumor antigen; 5) cryopreservation prior to armed normal orpatient ATC. OKT3×9184 armed normal ATC lyse PC-3, MCF-7 and SK-BR-3tumor targets. Increasing the arming dose of OKT3×9184 increases the %specific cytotoxicity directed at MCF-7, SK-BR-3 and PC-3 targets. Flowcytometry shows that OKT3×9184 binds to MCF-7, SK-BR-3 and PC-3 cells.Arming ATC with OKT3×Herc significantly enhances targeting of MCF-7,SK-BR-3 and PC-3 targets. Doses of OKT3×Herc and OKT3×9184 for armingATC are similar for targeting MCF-7 and PC-3 targets. Binding of ATCarmed with OKT3×9184 or OKT3×Herc to SK-BR-3 induces IFNγ secretion.OKT3×9184 or OKT3×Herc armed normal ATC are comparable in killing PC-3.Armed patient T cells remain cytotoxic to PC-3 after freeze/thaw.Cytotoxicity mediated by ATC armed with OKT3×Herc is not inhibited bysoluble Herceptin®. Clinical toxicities and efficacy were evaluated in aSCID mouse model showing that OKT3×anti-CEA armed ATC could prevent thegrowth of LS174T colon carcinoma cells in 10% of the SCID mice injectedin an Winn assay without any clinical toxicities when givensubcutaneously. Although there was no clinical benefit, I.V. tail veininjections and intraperitoneal injections of armed ATC did not causemorbidity or mortality in SCID mice. Cytokine secretion by normal ATCarmed with OKT3×9184 or OKT3×Herc is induced by binding to PC-3.Transgene cytokine and endogenous cytokine secretion can be reinduced bytargeting of IL-2 gene transduced-ATC. Clinical scale-up of armingprocess shows no differences in the arming doses needed for specificcytotoxicity in both normal and patient ATC. Anti-CD3/anti-CD28coactivated T cells from a normal can be armed with OKT3×9184 andexhibit specific cytotoxicity directed at MCF-7. Cryopreservation hadlittle affect on specific cytotoxicity directed at Her2⁺ MCF-7 or PC-3targets using both OKT3×9184 or OKT3×Herc. Cancer patients COACTS andATC armed with OKT3×9184 have comparable specific cytotoxicity activityagainst MCF-7 targets.

Example 1 Chemical Conjugation of OKT3×9184

FIG. 1 shows the steps for heteroconjugating OKT3 with the anti-tumorassociated antigen MAb (anti-TAA) (9184). OKT3 (1-5 mg) in 50 mM NaCl, 1mM EDTA, pH 8.0 is reacted with a 5 fold molar excess of Traut's reagent(2-iminothiolane HCl); (Pierce, Rockford, Ill.) at room temperature for1 hr (STEP, FIG. 1). 1-5 mgs of 9184 in 0.1 M sodium phosphate, 0.15 MNaCl at pH 7.2, is reacted, in a separate reaction, with a 4 fold Mexcess of sulphosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulpho-SMCC from Pierce,Rockford, Ill.) at room temperature for 1 hr (STEP 2). Both Mabs arepurified on PD-10 columns (Pharmacia, Uppsala, Sweden) in PBS to removeunbound crosslinker. The crosslinked Mabs are mixed immediately atequimolar ratios and conjugated at 4° C. overnight (STEP 3). Proteinquantitation is done with a BCA Protein Assay Kit (Pierce, Rockford,Ill.). The heteroconjugation product is analyzed by non-reducing SDS gelelectrophoresis using a 2-15% gradient gel (OWL Scientific, Woburn,Mass.). Coomassie blue staining is used to visualize proteins in thegel. Densitometric analysis is performed using the Gel Doc 1000 System(Bio-Rad, Hercules, Calif.). Densitometric quantitation of lane 5 of thegel showed ˜67% monomer, ˜23% dimer, and ˜10% multimer fractions (FIG.2). Lane 1, high molecular weight marker; lane 2, 10 μg of OKT3 wasloaded; lane 3, 10 μg of 9184 was loaded; lane 4, blank; lane 5, 30 μgof Her2Bi was loaded. The unconjugated OKT3 and 9184 (monomer), dimer ofOKT3×9184, and multimers of OKT3×9184 are indicated in lane 5.

Other anti-CD3 heteroconjugates are made with an irrelevantanti-prostate specific membrane antigen (anti-CD3×IG3), Herceptin®(anti-CD3×Herc), Rituxan (anti-CD3×Rit), T84.66 (anti-CD3×T84.66) areproduced using the same procedure.

Example 2 Rosetting of ATC armed with Her2Bi with MCF-7 or PC-3 Cells

All of the arming is always expressed as ng per million ATC using theentire heteroconjugate unless otherwise stated. Unarmed ATC (lower left,FIG. 3), ATC armed with 50 ng of Her2Bi (upper left), ATC armed with 50ng of irrelevant OKT3/IG3 (upper right), or ATC treated with a mixtureof 250 ng of nonconjugated OKT3+250 ng of non-conjugated 9184 (lowerright) are cultured with MCF-7 cells overnight at 37° C. An overnightculture at an effector-to-target (E/T) ratio from 90:1 to 3:1 wouldconsistently rosette with MCF-7 or PC-3 cells.

Example 3 Binding of Her2Bi to ATC

Phycoerythrin (PE)-conjugated goat anti-mouse IgG2a and IgG orfluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG1(Caltag Laboratories, Inc., Burlingame, Calif.) is used to detectbinding of Her2Bi. Binding is determined by incubating 10⁶ ATC with thesample at the stated concentration of Her2Bi for 1 hr at 4° C., washingtwice with PBS containing 1% BSA, and incubating for 30 mins in the darkwith PE or FITC conjugated goat-anti-mouse to determine the amounts ofOKT3 (IgG2a) or 9184 (IgG1) on the surface of the ATC. Binding of Her2Bito PC-3, MCF-7, and SK-BR-3 were determined by incubating 10⁶ MCF-7 orSK-BR-3 with 1μg BiAb for 1 hr at 4° C. The cells are washed andincubated with goat anti-mouse IgG1-FITC.

In order to quantitate binding, the mean fluorescence intensity (MFI) isdetermined for increasing doses of Her2Bi used to arm ATC (FIG. 4, PanelA). One million ATC were armed with 0, 0.5, 5, 50 and 500 ng of Her2Bi.Panel A shows that 9184 (an IgG1) portion of Her2Bi was detected inincreasing amounts on the surface of ATC with directly conjugated goatanti-mouse IgG allotype specific antibody. An arming dose of 0.5 ngcould still be detected about the background staining for the isotypecontrol.

Dual staining was performed using anti-IgG1 and anti-IgG2a specificreagents to demonstrate specific binding of the Her2Bi. In order tospecifically detect the IgG1 portion (9184) of Her2Bi, ATC armed with 50ng of Her2Bi are stained with goat anti-mouse IgG1-FITC for 9184. Todetect the IgG2a portion of Her2Bi (OKT3), armed ATC are stained withgoat anti-mouse IgG2a-PE for OKT3. The MFI for an arming dose of 5 ngwas 82 with 38% of the ATC being positive. At a dose of 50 ng, the MFIwas 223 with 93% being positive. Binding did not increase when thearming dose was further increased to 500 ng. Based on these data, 50 ngis selected as the saturating arming dose. The MFIs for 9184 and OKT3were 68 and 1050, respectively. Over 95% of ATC stained with IgG1-FITCand IgG2a-PE clearly indicated binding of Her2Bi.

Example 4 Detection of Her2/neu Receptors on MCF-7 and SK-BR-3 Cells

MCF-7 or SK-BR-3 are incubated with 1 μg Her2Bi or 9184, washed, andstained with IgG1-FITC. The histogram shows binding of IgG1 (9184portion of Her2Bi) to SK-BR-3 and MCF-7, respectively. Both 9184 andHer2Bi were detected with goat anti-mouse IgG1-PE with an MFI of 1133 on97% of SK-BR-3 cells which expresses high numbers of Her2/neu receptors(Panel D, FIG. 4). Irrelevant OKT3/IG3 did not bind to SK-BR-3 (notshown). MCF-7 cells (10⁶) were incubated with 1 μg Her2Bi or 1 μg of9184. The tumor cells were washed and stained with IgG1-FITC. Thehistograms show the binding of IgG1-FITC to MCF-7. 9184 and Her2Bi weredetected on only 64% and 48% of MCF-7 cells with MFIs of 28 and 35(Panel E) confirming the barely detectable expression of Her2/neureceptors on MCF-7 cells.¹⁵⁸

Example 5 The Development of Specific Cytotoxicity Directed at MCF-7

Cytotoxicity was measured in a ⁵¹ Cr-release assay. Tumor cells areplated in a flat-bottomed microtiter plate at a cell concentration of4×10⁴/well. The plates are incubated at 37° C. overnight, washed once,labeled with ⁵¹Cr (2 mCi/well) (Amersham, Arlington Heights, Ill.) for 4hrs at 37° C., washed 3 times and ATC are plated in triplicate at foureffector to target ratios (E/T). After overnight culture, 100 μl aliquotof the culture supernatant is removed for gamma counting on a COBRA-IIgamma counter (Packard, Downers Grove, Ill.). The % specific lysis=(cpmtest−cpm spontaneous)/(cpm max−cpm spontaneous)×100. Spontaneous releaseis determined by incubation of targets with media alone; and maximumrelease is determined by lysing the targets in 1% Triton-X 100 (SigmaChemical Co., St. Louis, Mo.).

To define the development of redirected cytotoxicity mediated by armedATC, fresh PBMC or ATC from the three normal subjects were armed with 50ng of Her2Bi and tested for cytotoxicity against MCF-7 cells on the daysindicated in FIG. 5. Each set of horizontal panels shows data for 3separate normal subjects. PBMC were tested on day 0 (D0) and ATC wereleft unarmed (•, no antibody) or armed with 50 ng of Her2Bi (▪) 50 ng ofOKT3×IG3 (▴), on day 4 (D4), day 6 (D6), day 8 (D8), day 13 or day 14(D13 or D14).

Lysis of MCF-7 cells by armed fresh (inactivated) PBMC prior to culturewas consistently <10% at all E/T and ATC armed with 50 ng ofTraut-crosslinked OKT3 or irrelevant OKT3×IG3 exhibited 30% specificlysis as all time points and E/T's tested. Arming after 6 days ofculture consistently enhanced specific cytotoxicity.

ATC armed with Her2Bi are so potent that they bind and kill MCF-7,breast cancer cell line that expresses very few Her2/neu receptors. Ourdata indicate that our new binding and cytotoxicity assays may be moresensitive than classic immunohistochemical staining for Her2/neu. MCF-7cells are consistently killed by Her2Bi armed ATC even though flowcytometry barely detects the presence of Her2/neu on the surface ofMCF-7 (FIG. 4, panel E). Our data suggest that even very low expressionof Her2/neu on prostate cancer cells may be targeted and lysed in vivo.

Example 6 Specific Cytotoxicity Directed at MCF-7 Increases with ArmingDoses and FIT

In order to determine the optimal arming dose range for Her2Bi, dosetitration studies are performed at E/T from 5:1 to 25:1. FIG. 6 showscomposite titration curves for unarmed ATC and ATC armed with 0.5, 5.0,and 50.0 ng of Her2Bi at E/T between 5 and 25. Each curve represents theinterpolated mean % (±SEM) specific cytotoxicity of 3 experimentsdirected at MCF-7 targets. Each dose titration curve shows theinterpolated mean of data using ATC from 3 normal subjects at theindicated E/T. Unarmed (▾) or ATC armed with 0.5 (), 5.0 (▪), and 50(♦). Increasing the arming doses of Her2Bi led to increasing mean %specific cytotoxicity. ATC armed with 25-500 ng of irrelevant OKT3×IG3or OKT3 exhibited % specific cytotoxicity similar to that of unarmed ATC(data not shown).

Example 7 Enriched T Cell Subsets were Tested to Determine the T CellSubsets for Specific Cytotoxicity

Dynabeads −450 CD4 (DYNAL, Lake Success, N.Y.) are used with DETACHaBeadmethodology for the positive selection of CD4 cells and the negativeselection of CD8 cells (CD4-depleted). The phenotype of the ATC and ATCsubsets after separation is determined by FACS analysis. TheCD4-selected cells were 99% CD4+ and the CD4− depleted cells were 94%CD8+. Unfractionated CD3+, CD4+ and CD8+ cells were armed with 50 ngHer2Bi and tested for their ability to mediate redirected cytotoxicityagainst MCF-7 cells (FIG. 7). At all E/T, the % specific cytotoxicityfor armed CD3+, CD4+, and CD8+ cells were consistently higher than theunarmed CD3, CD4, and CD8 populations and hierarchy of cytotoxicity wasCD8>CD3>CD4+ cells.

Example 8 Armed ATC Bind and Exhibit Cytotoxicity for More than 2 Days

To determine how long ATC remain armed and continue to kill tumor cells,flow cytometry and cytotoxicity assays are performed at 24-hr intervalsafter arming. ATC from a normal subject was armed with 0, 50 and 500 ngof Her2Bi. The Her2Bi is detected using goat anti-mouse IgG-PE (FIG. 8,Panel A). The results show that although there was a rapid decrease inbinding after 24 hrs, the remaining Her2Bi was stable and detectableafter 72 hrs. Her2Bi binding paralleled that of OKT3. These studies showthat hetero-conjugation did not impair binding of the anti-CD3 partnerin Her2Bi. ATC armed with as little as 5 ng Her2Bi exhibited 33±1% lysisof MCF-7 cells 24 hrs after arming. ATC armed with Her2Bi continued tokill MCF-7 targets up to 54 hrs and a % specific lysis of 35±4% was seenat an E/T of 24:1. At higher arming doses of 50 and 500 ng, % specificlysis was >48% with no decrease in cytotoxicity after 54 hrs. ATC armedwith 500 ng parental OKT3 or 9184 did not exhibit cytotoxicity abovebackground. These results show that Her2Bi redirected cytotoxicitypersists for 54 hrs.

Example 9 Enriched BiAb Activity in the Dimer-Enriched Fraction

OKT3×T94.66 (anti-CEA) heteroconjugates were produced to arm ATC to killCEA⁺ LS174T colon carcinoma cells. To confirm the fraction responsiblefor binding and targeting CEA, the dimer and multimer containingfractions are separated from the monomer fractions using a Sephacryl 300column and testing each fraction after adjusting all proteinconcentrations to the same for % specific cytotoxicity directed atLS174T E/T (FIG. 9). The solid squares show the dimer fraction; thesolid circles indicate the unfractionated material; the upward pointingsolid triangles indicate the monomer fraction; and the downward pointingsolid triangles indicate unarmed ATC. ATC were armed with 50 ng of eachfraction. The dimer-enriched fraction was enriched for cytotoxicityabove that seen for the unfractionated heteroconjugate whereas themonomer fraction is relatively depleted of cytotoxicity.

Example 10 Her2Bi Armed ATC from 10 Normal Control Subjects

FIG. 10 summarizes data from 10 normal subjects armed with 50 ng ofOKT3×9184 per 10⁶ ATC. At an E/T of 20:1, the mean increases in %specific lysis of MCF-7 cells by armed ATC from 10 normal subjects and 6patients were 59∀11% and 32∀9%, respectively. These means weresignificantly higher than unarmed ATC at an E/T of 20:1 (P<0.001 fornormal subjects, paired student's t test). Specific cytotoxicity was notdifferent from ATC alone when ATC were armed with 50 ng of unconjugatedOKT3, 50 ng of unconjugated 9184, or 50 ng of OKT3×IG3 (data not shown).

Example 11 In Vivo ATC Mediated Cytotoxicity

ATC from cancer patients that had been cryopreserved using control ratefreezing on the day of culture indicated on each panel (FIG. 11), werethawed and armed with Her2Bi. ATC were tested for cytotoxicity directedat MCF-7. The patients with renal cell carcinoma (3) had no priorchemotherapy whereas the patients with breast cancer (2) and lymphoma(1) had prior chemotherapy. The % specific cytotoxicity for 6 patientsincreased by 32±9%. Arming with Her2Bi significantly increased %specific cytotoxicity mediated by patient ATC (P<0.0004, pairedstudent's t test). The results indicate that there was no differencebetween those who received chemotherapy and those who did not receivechemotherapy.

Example 12 Cytokine Secretion Induced by Binding to SK-BR-3 Tumor Cells

Since secretion of interferon-γ (IFNγ), tumor necrosis factor-α (TNFα)and GM-CSF are hypothesized to provide anti-tumor effects, experimentswere conducted to test whether binding of armed ATC to SK-BR-3 wouldinduce IFNγ, TNFα and GM-CSF secretion (FIG. 12). Tumor cells wereplated at a concentration of 2×10⁵ per well in a 24-well plate andincubated overnight t 37° C. The next day unarmed and armed ATC(2×10⁶/well) were co-cultured with tumor cells (T) in a total volume of2 ml. Culture supernatants for each sample were pooled and tested forIFNγ, TNFα, and GM-CSF by ELISA (R&D Systems, Minneapolis, Minn.). Allvalues are reported in ug/m1/10⁶ ATC/24 hrs. Unarmed or ATC armed withdoses of irrelevant OKT3×IG3 up to 500 ng, or 9184 in the presence orabsence of tumor did not induce any IFNγ or TNFα (data not shown).Background was <5 pg. When ATC armed with Her2Bi (at both the 50 and 100ng doses) were co-cultured with SK-BR-3, the cultures markedly increasedtheir production of IFNγ, TNFα and GM-CSF above background. Stimulationof ATC with Trauts-crosslinked OKT3 control produced 110 ng of IFNγ inthe absence or presence of tumor and <50 pg of TNFα in the absence orpresence of SK-BR-3. Similar results were obtained using MCF-7 and PC-3.

Example 13 In Vitro Cytotoxicity Mediated Against Various Tumor CellLines

Arming of ATC or COACTS with Her2Bi significantly enhanced lysis of theHER2+ tumor targets MCF-7, SK-BR-3, and PC-3. We investigated the MCF-7breast carcinoma lines as a model target that expresses low numbers ofHER2 receptors (HER2r), the SK-BR-3 breast cancer line as a model targetthat expresses high numbers of HER2r, and the PC-3 prostate carcinomacell line (intermediate numbers of HER2r as the organ specific targetfor this clinical trial. The results using the MCF-7 model provide astrong rationale for targeting even weakly positive tumors using Her2Biarmed ATC. Binding studies show that PC-3 have more HER2 receptors ontheir surface than MCF-7 (FIG. 15). Her2Bi was found to remain on armedATC for over 72 hours and continue to mediate redirected cytotoxicityfor more than 54 hours.

Her2Bi armed ATC were tested against PC-3 (left panel) and MCF-7 (rightpanel) in FIG. 13. ATC were left unarmed (•), armed with 50 ng OKT3/IG3(▾), or Her2Bi (▪). The E/T titrations show that armed ATC are cytotoxicfor both PC-3 and MCF-7 carcinoma cell lines. At low E/T ratios, the %specific cytotoxicity appears to be higher for PC-3 than MCF-7 cells.These results are consistent with the flow cytometry findings that showPC-3 carcinoma cells have more Her2 receptors on their surface thanMCF-7 cells.

FIG. 14 indicates that increasing the arming dose of OKT3×9184 increasesthe % specific cytotoxicity directed at PC-3. ATC alone or ATC armedwith 0.1, 0.5, 1.0, 5, 50, and 500 ng of Her2Bi as indicated in FIG. 14and tested against PC-3. The irrelevant BiAb was OKT3×Rit. Cytotoxicitydirected at PC-3 was near optimal at a dose of 50 ng. Similar resultswere obtained on ATC from 3 normal subjects in similar titration curves.

FIG. 15 illustrates the results obtained using flow cytometry. The datashow that Her2Bi binds to PC-3 cells. PC-3, MCF-7 and SK-BR-3 weretreated with 1 μg of OKT3×9184 or OKT3 and subsequently stained withPE-conjugated goat anti-mouse IgG antibodies to determine the level ofexpression of Her2 receptors on PC-3 in comparison to MCF-7 (lowexpression) and SK-BR-3 (high expression). FIG. 15 shows clear bindingof OKT3×9184 to PC-3 and SK-BR-3 cells and the expected very low bindingto MCF-7.

Example 14 OKT3×Herc Cytotoxicity Directed at SK-BR-3 Targets

As described earlier in FIG. 1, heteroconjugating OKT3 with Herceptin®is conducted in an identical manner. OKT3 (2 mg) in 50 mM NaCl, 1 mMEDTA, pH 8.0 is reacted with a 5 fold molar excess of Traut's reagent.Herceptin (2 mgs) in 0.1 M sodium phosphate, 0.15 M NaCl at pH 7.2 isreacted with a 4 fold molar excess of Sulpho-SMCC at room temperaturefor 1 hr (STEP 2). Both monoclonal antibodies (Mabs) are purified onPD-10 columns in PBS to remove unbound crosslinker. The crosslinked Mabsare mixed immediately at equimolar ratios and conjugated at 4° C.overnight (STEP 3). BCA protein is quantitated and analyzed bynon-reducing SDS gel electrophoresis using a 2-15% gradient gel (FIG.16). Coomassie blue staining is used to visualize proteins in the gel.Lane 1, high molecular weight marker; lane 2, OKT3 (4 μg); lane 3, 9184(4 μg); lane 4, OKT3×9184 (4 pg); lane 5, OKT3×9184 (8 μg); lane 6, Herc(4 μg); lane 7, OKT3×Herc (4 μg); and lane 8, OKT3×Herc (8 μg). Theproportion of dimers in lanes 3,-4,6. and 7 as measured by densitometricquantitation was 27, 27, 21 and 21%, respectively.

FIG. 17 shows the ability of ATC from two normal subjects to lyseSK-BR-3 targets. ATC from normal (A) were tested unarmed; armed with 50ng of OKT3×Herc; armed with the irrelevant OKT3×4D4, a monoclonalantibody raised against prostate specific membrane antigen (gift fromAlton Boynton); or, a mix of 50 ng of unconjugated OKT3 and 50 ng ofunconjugated Herc. Normal (B) was tested in the same manner. Only theunarmed ATC and ATC armed with 50 ng of OKT3×Herc are shown.

Example 15 Comparative Arming Doses

In order to determine arming dose differences between OKT3×9184 andOKT3×Herc, ATC are armed with increasing doses of OKT3×Herc andOKT3×9184. Both BiAbs are effective in arming ATC to kill SK-BR-3targets (see FIG. 18). FIG. 18 shows that the OKT3×Herc may be slightlyless effective than OKT3×9184 at the 5 and 50 ng arming doses forspecific cytotoxicity directed at SK-BR-3 targets at the lower E:Tratios. These differences may be due to experimental variation in theseassays. The important observation is that arming at 50-500 ng/10⁶ ATCmarkedly enhances the ability of ATC to lyse SK-BR-3 targets.

Example 16 IFN Secretion by Armed ATC

Normal ATC arc armed with 50 or 100 ng of OKT3×9184 or OKT3×Hercrespectively and cocultured with SK-BR-3 targets at a E:T of 10:1 andculture supernatants were tested for IFNγ secretion after 24 hrs ofculture (FIG. 19). Arming with irrelevant OKT3×IG3 or a mixture ofOKT3+9184 or OKT3+Herc as control induced less than 200 pg ofIFNγ/ml/10⁶ ATC. At an arming dose of 100 ng for both BiAbs, there werecomparable induction of IFNγ secretion. At an arming dose of 50 ng, thebinding of ATC armed with OKT3×Herc induced less IFNγ than the bindingof ATC armed with OKT3×9184. However, this may be due to experimentaldifferences.

In order to determine how low an E:T would be effective in stimulatingIFNγ upon binding of ATC armed with 50 ng of OKT3×Herc, 24 hr culturesupernatants derived from armed ATC, cocultured with SK-BR-3 targets atE:T ratios of 5:1, 2.5:1, 1.25:1, and 0.625:1, were tested. E:T ratiosas low as 2.5:1 show an increase over background levels seen using noantibody (ATC alone), a mixed of soluble OKT3 and Herc, or arming withthe irrelevant OKT3×IG3 BiAb. Based on these titration experiments, allcytokine secretion studies were done with E/T ratios between 5:1 and10:1.

Example 17 Comparison of Cytolytic Capabilities of Her2Bi or OKT3×HercArmed Normal ATC Using PC-3 Targets

FIG. 20 shows a direct comparison between OKT3×9184 and OKT3×Herc atarming doses of 5, 50, and 500 ng. ATC armed with 500 ng of irrelevantOKT3×Rit is shown as a control at the highest E/T ratio. These data showthat both OKT3×9184 and OKT3×Herc exhibit similar trends in titrationfor PC-3 cells. In this experiment, the cytotoxicity curves for theOKT3×9184 dose titrations are higher than those seen for ATC armed withcomparable dose of OKT3×Herc.

Armed patient T cells remain cytotoxic to PC-3 after freeze/thaw.OKT3×9184 armed ATC from a patient with cancer were expanded for 14 daysafter anti-CD3 activation, cryopreserved, thawed, armed with 50 ng/10⁶ATC, and then tested for specific cytotoxicity (FIG. 21). ATC were armedwith 50 ng of OKT3×9184 (◯), of OKT3×Herc ( ) or OKT3×Rit (∇). A mix ofsoluble OKT3+9184 (▪) was also used as a control. These data show thatfrozen and thawed ATC can be armed and mediate cytotoxicity directed atPC-3.

Example 18 IFNγ ELISPOTS Produced by Normal ATC Armed with OKT3×9184Upon Binding with PC-3 Cells

In order to determine the frequency of the number of armed ATC thatproduce IFNγ when exposed to PC-3 cells bearing Her2/neu, ATC armed with50 ng/million of OKT3×9184 are placed onto plates containing 40,000 PC-3cells per flat bottomed microwell for 4 hrs at an effector to targetratio of 10:1. After 4 hrs, the effectors are removed and plated intoMillipore multiscreen nitrocellulose HA plates that are precoated withmonoclonal 7-B6-1 anti-human IFNγ antibodies. The plates are developedand the number of ELISPOTS are counted using a dissecting microscope.FIG. 22 summarizes the spots/million armed ATC for ATC alone (O), ATCmixed with PC-3 (PC-3), ATC restimulated with 50 ng of soluble OKT3(OKT3), ATC stimulated with 50 ng of OKT3 and PC-3 (OKT3+PC-3), ATCarmed with OKT3×9184 without tumor (Her2Bi), and ATC armed withOKT3×9184 mixed with PC-3 (Her2Bi+PC-3). Note the error bars indicate ±1SD of triplicate counts. There is a clear and brisk response of armedATC to engagement with PC-3 cells via the Her2Bi bridge.

Old and new lots of OKT3×9184 stable for at least 11 months. There wasno difference in the arming and specific cytotoxicity mediated by twobatches of OKT3×9184 produced Dec. 9, 1997 (old) and Nov. 5, 1998 (new).FIG. 24 shows ATC armed with 12.5, 25, and 50 ng of old or newOKT3×9184. The dose titration curves at each dose overlap.

FIG. 23 shows that cytotoxicity mediated by ATC armed with OKT3×Herc isnot inhibited by soluble Herceptin®. Increasing concentrations ofanti-Her2 antibody (0.5, 5, 50, 100, 500, 1000, 5000 ng of Herceptin®did not block cytotoxicity mediated by Her2i armed ATC until 1000 ng/mlwas reached. Therefore, circulating Herceptin® would not inhibit targetkilling by Her2Bi armed ATC. On the other hand, prebinding of tumortargets with Her2Bi prior to the addition of unarmed ATC did not formrosettes or enhance specific cytotoxicity (data not shown). In otherwords, pretreatment of a patient with Her2Bi would not provide enoughbinding for unarmed ATC to bind via CD3 to the tumor. The error barsindicate ±1 SD.

Example 19 In Vivo Efficacy and Cytotoxicity

Multiple intravenous injections or intraperitoneal injections of 20 to50×10⁶ ATC armed with 50 ng of OKT3×anti-CEA were not toxic to SCIDmice. Subcutaneous coinjections of armed ATC (20×10⁶) and CEA+LS174(1×10⁶) colon carcinoma cell line (Winn Assay) prevented tumorprogression and death in 40% of the mice that received armed ATC whereasonly 10% of the mice that receive ATC alone survived more than 100 days(FIG. 25). The SCID mice received 3 Gy of total body irradiation toeliminate NK cells to assure engraftment of tumor cells 1×10⁶ LS174T.All of the control SCID (tumor without ATC) mice died of tumorprogression by day 22 (tumor size >22 mm).

Example 20 Cytokine Secretion by Normal ATC Armed with Her2Bi orOKT3×Herc Induced by Binding to PC-3

Tumor cells (200,000) are plated as described supra and incubatedovernight. The next day unarmed or armed ATC (2×10⁶/well) areco-cultured with tumor cells (T) for 24 hours. Culture supernatants arepooled and tested for IFNγ, TNFα and GM-CSF (G) as described above. Theresults indicate that in the absence of tumor, ATC or ATC armed withOKT3 or OKT3/Rit, there were low levels of IFNγ produced. When normalATC armed with 50 ng of Her2Bi or OKT3×Herc were co-cultured with PC-3(FIG. 26), the ATC markedly increased their production of IFNγ, TNFα andGM-CSF (error bars indicate ±1 SD).

FIG. 27 shows the results for T cells from a patient that were armedwith Her2Bi or OKT3×Herc. ATC were grown from a blood sample after apatient with Ewing's Sarcoma had already undergone 5 cycles ofifosfamide and etoposide chemotherapy. Binding of Her2Bi armed ATC withPC-3 induced 5 marked fold increases in IFNγ and TNFα synthesis.Although GM-CSF synthesis in the absence of PC-3 was higher, there was a2 fold increase in GM-CSF production by patient ATC. These data showthat ATC from patients who have received multiple cycles of chemotherapyare still capable of producing cytokines after arming with Her2Bi.

In retroviral gene transduction of ATC, the results indicate that bothIL-2 transgene and endogenous cytokine IFNγ levels had become quiescenta week or more after transduction of the retroviral vector containingIL-2. However, exposing OKT3×anti-CEA (T84.66) armed ATC to CEA+LS174Tinducted secretion of IL-2 and IFNγ.¹⁵⁷ Altogether, these data show thatthe triggering of cytokine secreting occurs using different tumorsbearing Hcr2 receptors as well as in a different model using a differentBiAb and colon carcinoma cell target.

Example 21 Clinical Scale-Up of Arming Doses Needed for SpecificCytotoxicity

Arming 1 billion ATC was performed at a dose of 0 ng and thecytotoxicity directed at MCF-7 were compared to a series of dosetitration curves ranging from 0.1 to 500 ng. FIG. 28 shows the data forthe normal (NL) and FIG. 29 shows the data for the patient (PT).

One billion ATC were armed with 50 ng/million ATC by adding 50micrograms to one billion ATC in an Ethox 3 L bag in plasmanate. Thedoses used to arm ATC in tubes are indicated in figure legend.

Cryopreservation had little affect on specific cytotoxicity directed atHer2+ MCF-7 targets. FIG. 30 shows the two aliquots are comparable infunctional cytotoxic activity. In order to test the effects offreezing/thawing on redirected cytotoxicity, an aliquot of normal ATCwas compared to aliquot of the same ATC that were cryopreserved, thawed,and then armed with 15 or 30 ng of Her2Bi, respectively. The right panelof FIG. 30 shows specific lysis for ATC that were frozen, thawed andthen armed with OKT3×Herc.

Example 22 Arming of Anti-CD3/Anti-CD28 Coactivated T Cells (COACTS)with Her2Bi (OKT3×9184 or OKT3×Herc)

COACTS were left unarmed or armed with 50 ng of OKT3×9184, 50 ng of 9184alone, or 50 ng of OKT3×IG3 and tested at the indicated E/T ratios forcytotoxicity directed at MCF-7. FIG. 31 shows that COACTS can be armedand lyse MCF-7 targets.

Cancer patients COACTS and ATC armed with OKT3×9184 have comparablespecific cytotoxicity activity against MCF-7 targets. COACTS and ATCwere produced over a 14 day culture period from the same blood samplesfrom three cancer patients. Armed COACTS or ATC from each patient wastested against MCF-7 targets. Panel P1, P2, and P3 of FIG. 32 show theability of unarmed and armed COACTS and ATC to mediate specificcytotoxicity.

A phase I dose-escalation study, was completed, using infusions ofautologous ex vivo expanded COACTS for the treatment of refractorycancer patients.⁹⁵ The technical limits of ex vivo COACTS expansion, thein vivo localization and trafficking of COACTS, and immune effectsinduced by COACTS infusions in the patients were evaluated. Infusions ofCOACTS were safe, induced detectable, serum levels IFNγ, GM-CSF, andTNFα, and significantly enhanced the ability of freshly isolated PBMC tosecrete IFNγ and GM-CSF upon in vitro anti-CD3/anti-CD28 costimulation.These data suggest that the immune systems of these patients weremodulated by COACTS infusions. Follow-up studies are in progress toevaluate COACTS in combination with chemotherapy and biologic responsemodifiers.

Example 23 BiAb Targeting Reactivation of Gene-Transduced T CellsEnhances both Endogenous Cytokine Production and Secretion of theTransgene Cytokine Product Expression

In order to circumvent transgene downregulation, OKT3×T84.66(anti-CD3×anti-carcinoembryonic antigen) was used to bind and retargetIL-1α gene transduced ATC.¹⁵⁷ ATC had been modified with a IL-1αretroviral vector and armed with anti-CD3×T84.66 and mixed with LS174T(CEA+ colon carcinoma line). Binding of armed ATC to LS174T cells led tothe induction of both endogenous IFNγ secretion and IL-1α secretion.These data further support the use of armed ATC.

Example 24 Treatment of Human Patient with Armed ATC Specific forPancreatic Cancer

The patient (AR), with pancreatic cancer, was leukopheresed for astarting population of 9.8×10⁹ mononuclear cells with 58% CD3⁺ cells,43% CD4⁺, and 14% CD8⁺ cells. The ATC were harvested and armed 10 dayslater with a total harvest of 61.8×10⁹ ATC consisting of 94% CD3⁺, 83%CD4⁺, and 12% CD8⁺ cells. The patient has received multiple doses of2.5×10⁹ ATC armed with a dose of 50 ng OKT3×Herceptin bispecificantibody over two weeks without any dose limiting toxicities.

Table 3 below shows a summary of male patients with hormone refractoryprostate cancer (HRPC) who were/are being treated with armed ATC andtheir clinical toxicities using OKT3×9184. Although, the bispecificantibody contains another anti-HER2/neu monoclonal antibody, thetoxicities are similar.

TABLE 3 Summary of Treatment of Patients HRPC Prostate Cancer SurvivalDose Patient Treatment Disease Course Delivered ≧Grade 3 Initials MRU#Age Started Status Completed (×10⁹) Toxicity 1 R G 975929 65 Aug. 05,2001 Oct. 13, 2001 Yes 20 Chills Died of Progression 2 T N 0982204 81Sep. 12, 2001 Died of Yes 20 Chills Progression 3 P P 523902 75 Feb. 8,2002 Alive, No Yes 20 Chills response so far 4 E M 1008188 85 May 7,2002 Alive, Not 6^(th) Infusion 12 Chills evaluable

It is clear that there are no dose limiting toxicities at the doses thathave been given to the 4 patients with prostate cancer (Table I), 1patient with Stage II breast cancer, and the first patient withpancreatic cancer. All of the infusions were completed on an outpatientbasis.

Table 4 summarizes the expansion of ATC prior to arming and phenotypingdata of the product that was infused into the men with hormonerefractory prostate cancer.

TABLE 4 Summary of Product Expansion (Cells × 10⁹) Starting Days of %CD3 % CD4 % CD8 Total CD3 CD4 CD8 Pt MNC Culture Pre Pre Pre HarvestViability Harvest Harvest Harvest 1 9.4 11 15 8 7 40 95 38 34 2.8 2 14.716 37 28 9 32 90 31 28 2.2 3 15.1 13 36 24 12 35 95 34.3 20 12 4 15.3 1417 — — 38 95 35.3 28.5 8.36

Example 25 Confirmation of Specific Cytotoxicity Directed at HER2/neupositive Pancreatic Cell Lines Mediated by ATC from an Additional NormalSubject and Two Patients with Cancer

Additional data were obtained using ATC armed with 50 ng ofOKT3×Herceptin per million ATC. The FG pancreatic cell line was platedthe night before, radiolabeled with ⁵¹Cr, and unarmed ATC, ATC armedwith OKT3×Herceptin or ATC armed with irrelevant BiAb OKT3×Rituxan wereplated and ⁵¹ Cr release was assessed after 18 hrs of co-culture.Specific cytotoxicity for one normal subject and two patients withprostate cancer at effector to target ratios from 3.13 to 25.00 areshown in FIG. 33. Arming of the ATC from the two patients and normalclearly augmented specific cytotoxicity at all effector to target ratios(E:T).

In order to confirm cytotoxicity in a second pancreatic cell line, anadditional cell line, MIA was purchased from ATCC and tested in the sameassay. FIG. 34 shows that armed ATC were able to kill MIA targets.Western blotting of the MIA cell line confirmed the expression ofHER2/neu receptors on the cell line.

Example 26 ATC Armed with OKT3×Herceptin can Kill Multiple Times

In order to determine whether armed ATC could kill tumor cells multipletimes, a series of parallel cultures that contained unarmed, armed withHER2Bi, and armed with OKT3×Rituxan, was set up. SK-BR-3 breast cancertarget cells were plated overnight prior to each of the cytotoxicityassays. Before arming of the ATC, the ATC were marked with 5(6)carboxyfluorescein diacetate N-succinimidyl ester (CFDA-SE) dye so thatthe dye could be used to evaluate the number of cell divisions thatoccur and be detected by gating for green fluorescence using flowcytometry. Effector: Target ratios ranging from 2.5 to 10:1 were setupon the day of the cytotoxicity assay by performing cold counts of theplated target cells after each radiolabeling. This maneuver accounts forcell losses and provides the actual number of targets in each well priorto the addition of effector T cells. Panel A of FIG. 35 shows the 1^(st)cytotoxicity assay mediated by an aliquot of armed T cells and analiquot of unarmed ATC tested at time zero. After 48 hrs of incubationwith the first set of targets, the unarmed and armed ATC were harvestedand aliquots of each were replated onto a second set of targets for asecond culture and 2^(nd) cytotoxicity assay at 45 hrs. After thereplated unarmed (ATC) or armed ATC (aATC) were co-cultured with SK-BR-3between 48 hrs and 96 hrs, the unarmed and armed ATC were harvested andaliquots of each were replated onto a third set of targets for a thirdculture and a 3^(rd) cytotoxicity assay at 96 hrs. Finally, the unarmedand armed ATC that had been co-cultured with SK-BR-3 from 96 hrs to 215hrs were harvested and aliquots of each were replated onto a fourth setof targets in a 4^(th) cytotoxicity assay at 215 hrs. Since repeatedcytotoxicity was observed, the data show that the ATC were still “armed”with OKT3×Herceptin. Flow cytometry on the armed ATC at 213 hrs clearlyshowed persistence of OKT3 (murine IgG2a) on the surface of the ATC.Viability was consistently greater than 80% by propidium iodide and bytrypan blue exclusion.

Example 27 Cell Division Occurs in the ATC Population MediatingCytotoxicity

The experiment shown in FIG. 35, was conducted to determine whether thecells would divide after binding to HER2/neu antigen on the surface ofSK-BR-3 target cells. The flow cytometry data in FIG. 36, showed thatthere was evidence for several cell divisions during the multiple roundsof tumor antigen exposure as exhibited by the detection of cellsexhibiting decreased amounts of CFDA-SE fluorescence. Panels A and Bshow armed ATC 3 hrs and 48 hrs after mixing of the armed ATC withSK-BR-3 targets, Panels C and D show unarmed ATC at 3 hrs and 48 hrsafter mixing with SK-BR-3 targets. Panels E and F show armed ATC alonewithout exposure to targets. Panel B shows that nearly all of thepopulation has shifted downwards from the mean fluorescence intensity(MFI) of the original peaks of MFI's 30 and 18 on Panel A compared topeaks with MFI's of 30, ˜20, ˜10, and ˜5 on Panel B. Unarmed ATC (PanelD) mixed with targets have divided at roughly the doubling time seen forATC in culture with nearly all of the cells synchronized at point 2.Armed ATC in Panel F have started to divide and there is a largepopulation of cells that have divided (MFI ˜20) a smaller populationthat divided twice at a MFI of ˜9. In contrast, unarmed ATC in thepresence of SK-BR-3 do not proliferate, and armed ATC without targetsexhibit some proliferative shifts as a result of “low” level activationinduced by the arming process with a small population with a MFI of ˜35representing cells that had not divided. It is anticipated that armingprovides a low level restimulation to ATC. In summary, Panel B witharmed and targeted ATC shows 4 cell populations identified by the arrowsthat originated from populations #1 and #2 in Panel A.

Example 28 CD4⁺ or CDS⁺ T Cell Subsets Preferentially Proliferate

All of the ATC, were marked with CFDA-SE prior to arming with HER2Bi orirrelevant (control) OKT3×Rituxan, phycoerythrin labeled monoclonalantibodies directed at human CD4⁺ and CD8⁺ were used to determine theproportion of CD4+ and CD8+ cells that occurred within the wholepopulation of CFDA-SE positive ATC.

FIG. 36 presents a combination overlay from another experiment thatshows the numbers of CFDA-SE⁺ cells within the CD4⁺ or CD8⁺ subsets thathad been armed with OKT3×Herceptin, OKT3×Rituxan, or left unarmed. Thekey point is that the CD4⁺ cell population showed evidence of celldivision with a very distinct population of cells that had divided andshowed reduce intensity whereas the CD4⁺ cells armed with OKT3×Rituxanand unarmed ATC did not show as many cells that had divided andexhibited half as much staining intensity.

Example 29

No. Armed ATC Abs Count of spiked spiked % of WBC Gate ATC FIG. 6 Panel200,000 4.70 131  B 100,000 2.00 53 C 50,000 1.19 27 D 25,000 0.69 18 E12,500 0.40  11? F

Trafficking and Detection of Circulating Armed ATC after Infusion.

The survival or trafficking of circulating armed ATC after infusion weremonitored in patients using a newly developed flow cytometry assay todetect IgG2a bearing armed ATC. In order to determine what thesensitivity of flow cytometry for detecting T cells bearing membranebound IgG2a (the OKT3 portion of the BiAb or OKT3 alone), increasingknown amounts of ATC armed with 50 ng/million ATC, were added to freshheparinized blood and tested the whole blood for cells bearing mouseIgG2a with a PE conjugated polyclonal goat anti mouse-IgG2a antibody.

The six panels of FIG. 37 show that as few as 12,500 armed ATC per mlmixed with 5.58 million normal white cells in the gate (total WBC 5.58×10³/mm³from the CBC) can be detected by flow cytometry using thismethod.

In FIG. 37, Panel F, the data show that as low as 0.4% ATC armed withOKT3×Herceptin/million could be detected in whole blood. Based on thesesensitivity studies, we tested several random samples of blood andphenotyped the blood for murine IgG_(2a) bearing T cells. Up to 1.5%positive cells in the WBC gate (corrected for isotype control) weredetected in the peripheral blood of Patient MRU #523902 up to 8 daysafter his sixth infusion of armed ATC.

Example 30 In Vivo Survival of Armed ATC Following the First Infusion ofArmed ATC

Based on the detection of persistent IgG_(2a) bearing cells, a kineticstudy to determine the survival of armed ATC following the firstinfusion of armed ATC was performed. Phenotyping on the peripheral bloodwas obtained Pre, 10 mins, 30 mins, 1 hr, 4 hrs, 10 hrs, and 24 hrsafter an infusion of 2.0×10⁹ OKT3×9184 (anti-HER2/neu, a gift fromNexell) armed ATC. The kinetic study showed that armed ATC can bedetected at approximately as 1% of the total WBC gate as soon as 30 minsafter infusion. The armed ATC could not be detected around 4 hrs, butdetection and persistence in the patient's circulation occurred between10 hrs and 24 hrs after the infusion.

Example 31 Long-term Survival of Engrafted Human T Cells in Beige/SCIDMice

Follow-up studies on mice engrafted with human T cells went intoremission after 11 or 12 intra-tumoral injections of 50×10⁶ armed ATC.Phenotyping of the spleen and bone marrow revealed 5.5% CD3⁺ cells. Thecontrol mice had no detectable human CD3⁺ cells. The data show that thedirectly injected armed ATC, killed tumor cells and outnumbered thetotal number of injected armed ATC (600 million). The increase in numberof in vivo armed ATC is due to the fact that armed ATC divide at leastonce or more after engaging the tumor in vivo. It is important to notethat no IL-2 was given to the mice after their tumors regressed.

Example 32 Trafficking of ATC In Vivo and Accumulation at Tumor Sites

Blood phenotyping and biopsies of accessible metastatic lesions areobtained. The biopsied tissue is used in indirect immunofluorescenceassays using anti-IgG_(2a) antibodies to detect the OKT3 component ofOKT3×Herceptin®. The clinical protocol incorporates biopsies when theyare obtained. For example, peripheral blood mononuclear cells (PBMCs)can be obtained from a subject and isolated by density gradientcentrifugation, e.g., with Ficoll/Hypaque. Specific cell populations canbe depleted or enriched using standard methods. For example,monocytes/macrophages can be isolated by adherence on plastic. T cellsor B cells can be enriched or depleted, for example, by positive and/ornegative selection using antibodies to T cell or B cell surface markers,for example by incubating cells with a specific primary monoclonalantibody (mAb), followed by isolation of cells that bind the mAb usingmagnetic beads coated with a secondary antibody that binds the primarymAb. Peripheral blood or bone marrow derived hematopoietic stem cellscan be isolated by similar techniques using stem cell-specific mAbs(e.g., anti-CD34 mAbs). Specific cell populations can also be isolatedby fluorescence activated cell sorting according to standard methods.Monoclonal antibodies to cell-specific surface markers known in the artand many are commercially available.

Example 33 Prevention of Tumor Re-Growth

To prevent tumor growth, irradiated female Beige/SCID mice (the kindgift of Dr. Ray Frackelton) were co-injected subcutaneously in the hipwith 10⁶ PC-3 tumor cells and either: IL-2 alone, 10⁷ unarmed ATC andIL-2, 10⁷ armed ATC and IL-2, or 2×10⁷ armed ATC and IL-2. IL-2 wasgiven in the dose of 10 IU/g bodyweight. 50 ng of OKT3×Herceptin BiAbwas used to arm ATC. Once injected, mice were given no further treatmentbut were monitored for weight and length by width tumor dimensions everytwo to four days. Length by width dimensions were used to calculatetumor volume with a standard hemi-elipsoid formula: (length×width²)/2.Animals with 864 mm³ (12×12 mm) tumors were euthanized. Mice weremonitored continuously from first sign of tumor development through the105^(th) day following initial co-injection.

In an attempt to prevent established tumors in the Beige/SCID mousemodel, irradiated and non-irradiated females were injectedsubcutaneously in the hip with 10⁶ PC-3 tumor cells and allowed todevelop 62.5 mm³ (5×5 mm) tumors. Mice were given direct intratumoralinjections twice per week with either: IL-2 alone, 10⁷ armed ATC andIL-2, or 5×10⁷ armed ATC and IL-2. A 10 IU/g bodyweight dose of IL-2 wasmaintained. 50 ng of OKT3×Herceptin BiAb was used to arm ATC. As in thepreviously described Winn (or co-injection) assay, mice wereindividually tracked for length by width tumor dimensions and animalweight every two to four days. Mice with 864 mm³ (12×12 mm) tumors weresacrificed. Mice were monitored continuously until tumor remissions wereinduced, then periodically thereafter to track relapse.

Statistical Analysis

Specific cytotoxicity was calculated as the mean of triplicatewells±standard deviation using Excel (Microsoft Office). ELISA valuesfor cytokine and chemokine secretion were calculated as the mean ofduplicate wells using Excel. All graphical representations of in vitrodata were created using SigmaPlot (Jandel Corporation, San Rafael,Calif.).

For all animal experiments, Kaplan-Meier curves were generated. Tumorgrowth delay models were also generated, testing statistical differencesbetween the growth curves of each group with the non-parametricKruskal-Wallis test followed by Dunn's multiple comparisons test. Allstatistical analyses were performed using the Prism statistical program(GraphPad, San Diego, Calif.).

Flow Cytometry

Following tumor relapse in two animals that had undergone remission andtwo untreated, tumor-free control mice, bone marrow, spleen, andperipheral blood samples were harvested. Human CD3, CD4, CD8, CD16,CD45RO, CD45RA, and CD56 expression were evaluated by flow cytometry ona FACSCalibur System (BD Biosciences, San Jose, Calif.).

Results Armed Activated T-Cells Bind and Lyse HER2⁺ Tumor Cells

OKT3×Herceptin BiAb armed ATC exhibit enhanced specificity over unarmedor irrelevant (OKT3×Rituxan) BiAb armed ATC, resulting in high levels ofPC-3 prostate cancer cell lysis. Using an irrelevant BiAb to crosslinkCD3 proteins of an ATC without engaging tumor antigen fails to inducecytotoxicity above the level of unarmed ATC in the presence of tumortargets.

BiAb doses between 5 and 500 ng per 10⁶ ATC can double to triple the %specific cytotoxicity observed with unarmed T-cells (FIG. 38). BiAbdoses of 5 to 500 ng are optimal to redirect cytotoxicity of all ATC,since smaller doses of BiAb result in reduced levels of killing andaddition of more BiAb does not increase tumor cell lysis. Arming withBiAb enhances cytokine and chemokine secretion by activated T-cells.

Since GM-CSF, TNF-α and IFN-γ are known to produce anti-tumor effects(ELISA assays booklets, other reference), cell culture supernatants weremeasured after overnight incubation at 37° C. at a 10:1 E/T ratio. ArmedATC exhibit a distinct increase in GM-CSF, TNF-α and IFN-γ when comparedwith irrelevant armed or activated T-cells (FIG. 39). MIP-1α and RANTESchemokines may improve trafficking of armed ATC to tumor sites. For thisreason, cell culture supernatants were tested by ELISA to quantifychemokine secretion. Elevated levels of MIP-1α and RANTES chemokinesecretion were seen from armed ATC exposed to PC-3 tumor targets. As wascomparable with cytokine secretion, irrelevant armed or ATC alone showedmuch lower levels of chemokine secretion.

Example 34 Co-Injection of Armed Activated T-cells and Prostate CancerCells can Prevent Tumors in Mice

Control mice receiving only PC-3 tumor cells and 10 IU/g IL-2 were allsacrificed due to tumor burden by day 70. Three (n=13) mice receivingonly 10 ATC for every PC-3 tumor target (dose of 10⁷ ATC with IL-2)never developed tumor, while a fourth mouse was still alive with reducedtumor burden of 108 mm³ (6×6 mm) by day 105. Co-injecting with 10 armedATC per target (dose of 10⁷ ATC with IL-2) prevented tumors in 6 of 12mice. A seventh mouse was still alive by day 105 with reduced tumorburden of 108 mm³ (6×6 mm). The highest dose of armed ATC (2×10⁷ ATCwith IL-2) co-injected with PC-3 targets was sufficient to completelyprevent tumor development in the entire group (n=7), p=0.00005. TheKaplan-Meier survival curves of FIG. 40 represent the proportion ofmice, which have no subcutaneous tumor burden after co-injection.

FIG. 41 represents the relative efficacy of unarmed and armed ATC indelaying tumor growth among the groups of mice. A high dose of 2×10⁷armed ATC can completely prevent tumor growth in all mice by day 105,and is statistically significant when compared with the control(p<0.001). 50% fewer armed or unarmed ATC (10⁷) only delay tumor growthabove the rate of the control (p<0.01).

Example 35 Treating Established Tumors with Armed Activated T-Cells canEradicate Tumors in Mice

Once subcutaneous hip tumors reached a volume of 62.5 mm³ (5×5 mm),direct tumor injections were initiated. Throughout the injectionschedule (twice per week until remission was induced) established tumorsin both treatment groups, 10⁷ and 2×10⁷ aATC plus 10 IU/g IL-2, andcontrol mice, only 10 IU/g IL-2, were observed to continue growing.While all control mice were sacrificed due to tumor volumes of 864 mm³within six weeks of injection with tumor cells, remissions in bothtreatment groups were observed. Tumor growth in mice given the low dosetreatment of 10⁷ ATC gave similar results as compared to control mice(FIG. 42), with the exception of a single mouse whose tumor volumereached 666 mm³ before gradually decreasing to 224 mm³ and disappearing4 days later. This mouse was given 6 weeks of treatment only andsubsequently maintained tumor remission for 7 weeks without furthertreatment or IL-2, at which time relapse occurred and the mouse wassacrificed. Treating mice with 5×10⁷ ATC appeared to improve survivalwithin the first six weeks of treatment, when compared with low dose ATCor IL-2 treatment. In addition, similar remission results were observedin two mice given high dose aATC after 6 weeks of treatment. Two micewith tumor volumes approaching 864 mm³ simply “lost” their tumors within2-4 days and maintained tumor remission for 12 weeks without furthertreatment or IL-2. Although these 2 mice relapsed after 12 weeks andsurvival curves were not statistically significant (p=0.195), theanimals were sacrificed for engraftment analyses by flow cytometry.

Tumor growth delay analysis revealed only the highest dose of armed ATCwas effective when compared with the control, although the data were notstatistically significant (p=0.0636).

Example 36 Long-Term Human T-Cell Engraftment is Indicated with TumorRemission

Two female beige/SCID mice receiving no injections of tumor, ATCtreatment, or IL-2 were used as negative controls to evaluate humanT-cell engraftment in treated female beige/SCID mice. No detectable(<1%) human lymphocytes were observed in the bone marrow, spleen, andperipheral blood of both control mice.

Flow cytometry of the first treated mouse (5×10⁷ ATC) revealedapproximately 3% CD45RA+ and 1% CD45RO+ human cells in the bone marrow,nearly 5% CD4+ and possible 5% CD3+, CD45RA and RO+ populations in theperipheral blood, and no detectable human cells in the spleen. Resultsfrom the second treated mouse (same dose) revealed approximately 2.5%CD4+, 2% CD8+, and up to 2.5% CD45RA and RO+ populations in the bonemarrow, approximately 4% of each CD45RA and RO+, up to 3% CD8+, and 5.5%CD3+ populations in the spleen, and no detectable human cells in theperipheral blood.

Example 37 Phenotyping of Armed ATC to Determine Survival andTrafficking

In order to determine the kinetics of armed ATC after infusions, bloodis drawn for phenotyping before the 1^(st) infusion and 30 mins, 1 hr, 4hrs, 8 hrs, 24 hrs, 48 hrs, and 72 hrs (just prior to the 2nd infusion).Based on the kinetics that are observed, additional studies are done todefine the decay rate or accumulation of armed ATC during the multipleinfusions. After the 1^(st) infusion, samples are tested just prior toand 1 hr after each infusion. PE conjugated polyclonal goat anti-mouseIgG2a is used to detect OKT3 monomers or OKT3 dimers or multimers on thesurface of ATC. These studies will be coordinated with the ¹¹¹Indiumlabeled trafficking studies.

Clinical Correlates/Analyses.

The number of IFN ELI SPOTS from fresh unstimulated PBMC from men beforeand after infusions of armed ATC at the various time points arecompared. The number of IFNγ ELISPOTS at each time point are comparedwith the pretreatment number of ELISPOTS to look for differences in theprecursor frequency. The tests applied will determine whether: 1)baseline IFNγ ELISPOTS responses of men with HRPC without stimulationare different than normal age-matched men; 2) the induction of IFNELISPOTS responses is different in the same men before and afterinfusions of armed ATC and how long they remain elevated; 3) theresponses are specific or nonspecific to HER2 receptors; 4) thecirculating amount of HER2 receptors decreases as a result of a clinicalresponse; and 5) each of the immunologic or serum assays will becollated on a laboratory database and correlated with the clinicalresponse database to look for in vivo/in vitro correlates.

Example 38 Comparison of Immunologic Changes Between Baseline and EachPre-Determined Time Point after Receiving Armed ATC Treatment

A total of 28 patients are used to detect a difference of about 0.53standard deviation in proliferation, in ELISPOTS, or in the amount ofcytokine produced between baseline and after armed ATC treatment at eachtime point. A total of 28 patients provide a two-sided 95% confidenceinterval for the estimate of the proportion change in particularphenotype within ±11% if the true change in this population is 10%.Paired t-test or Wilcoxon signed rank test is used to compare thedifference between baseline and after armed ATC treatment inproliferation, ELISPOTS, and the amount of cytokine produced to identifythe time point when the change becomes significant. To examine the timeeffect on these immune responses, data from different study time point(including baseline) is analyzed using mixed model for repeatedmeasurements data.

The number of IFNγ ELISPOTS from fresh unstimulated PBMC from men beforeand after infusions of armed ATC at the various time points is comparedto test if in vivo lysis of tumors by the armed ATC induce memory Tcells capable of responding to PC-3 when exposed in vitro to tumor (arecall response) and to look for differences in the precursor frequency.Paired t-test or Wilcoxon signed rank test is used in these comparisons.Similarly, anti-CD3 stimulated cells will be compared before and afterimmunotherapy. Further comparisons will be made after stimulations withHER2+ and HER2− cell lines. The results from these stimulations will benormalized to the responses of the PBMC from age-matched men ascontrols. The means, medians and standard deviations are calculated forthe number of IFNγ ELISPOTS. Student's t-test are used to examinewhether the number of IFNγ ELISPOTS at baseline of men with HRPC withoutstimulation are different from normal age-matched men. Mean changes inthe induction of IFNγ ELISPOTS responses before and after immunotherapyare plotted over time to explore how long they can remain elevated.Mixed model for repeated measurements data is used to examine the timeeffect on the induction of IFNγ ELISPOTS in this study population. Mixedmodel for repeated measurements data is used to examine the time effecton the circulating amount of HER2 receptors. Logistic regression modeland Cox's proportional hazard regression model is used to evaluate eachof the in vivo or in vitro immunological or serum assays (e.g. HAMA) aspredictors of clinical responses (complete response/partial response)and progression-free survival, respectively.

Example 39 Multiple Exposure of the Anti-CD3 Activated Polyclonal T CellPopulation Induces the Development of HER2/neu Specific T Cell Clones

The data shown in FIG. 44 show the number of IFN gamma ELISPOTS from ATC(unarmed activated T cells that had been exposed 3 times to SK-BR-3),unarmed ATC that were exposed to a human EBV-driven B cell lines (a Bcell line would not express HER2/neu receptors; only the final time),and aATC (armed ATC that were exposed to SK-BR-3 three times and thenexposed a fourth time in this assay). The assay was performed on day 20after arming. No additional arming was performed from the initial armingwith 50 ng/million. Without wishing to be bound by theory,subpopulations of armed ATC were primed to HER2/neu and have becomememory cells as measured by their ability to respond vigorously torechallenge to HER2/neu antigen on the SK-BR-3 cells. Furthermore, theresults suggest that multiple exposure of the anti-CD3 activatedpolyclonal T cell population has selected or induced the development ofHER2/neu specific T cell clones.

This may be a new method for producing antigen specific clones directedat a specific tumor antigen as well as antigens on the tumor that areyet undefined and unknown. One advantage of this type of immunization isthat it would immunize the patient with his/her own antigens without anactual definition. This could happen in vivo as well as in our in vitromodel.

A new concept: Since there are no dendritic cells in the culture system,one very real possibility is that activated T cells can act professionalantigen presenting cells since they upregulate class II upon activationand may act together with the crosslinked tumor antigen in the presenceof cytokine and chemokines producted by the reactivation process toinduce antigen specific CTL. Class II upregulation may provide thenecessary help from CD4 helper cells in the polyclonal mixed to providethe signals needed to induce antigen-specific CTL. Our next experimentswill use dendritic cells and purified activated T cell subsets toaddress the question.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

The following references are herein incorporated in their entirety.

REFERENCES

-   1. Grimm, E. A., A. Mazumder, H. Z. Zhang, and S. A.    Rosenberg. 1982. J. Exp. Med. 155:1823-1841.-   2. Grimm, E. A., K. M. Ramsey, A. Mazumder, D. J. Wilson, J. Y.    Djeu, and S. A. Rosenberg. 1983. J. Exp. Med. 157:884-897.-   3. Hersey, P., G. Bindon, A. Edwards, E. Murray, G. Phillips,    and W. H. McCarthy. 1981. Int. T. Cancer 28:685-703.-   4. Lotze, M. T., E. A. Grimm, A. Mazumder, J. L. Strausser,    and S. A. Rosenberg. 1981. Cancer Res. 41:4420-4425.-   5. Lanier, L. L., A. M. Le, J. H. Phillips, N. L. Warner, and G. F.    Babcock. 1983. J. Immunol. 131:1789-1796.-   6. Hercend, T., J. D. Griffin, A. Bensussan, R. E. Schmidt, M. A.    Edson, A. Brennan, C. Murray, J. F. Daley, S. F. Schlossman, and J.    Ritz. 1985.. J. Clin. Invest. 75:932-943.-   7. Lotzova, E. and R. B. Herberman. Immunobiology of natural killer    cells. CRC Press, Boca Raton.-   8. Lotzova, E. and K. B. McCredie. 1978. Cancer Immunol. Immunother.    4:215.-   9. Rosenberg, S. A., M. T. Lotze, L. M. Muul, A. E. Chang, F. P.    Avis, S. Leitman, W. M. Linehan, C. N. Robertson, R. E. Lee, J. T.    Rubin, C. A. Seipp, C. G. Simpson, and D. E. White. 1987. N.    Engl. J. Med. 316:889-897.-   10. Mule, J. J., S. Shu, and S. A. Rosenberg. 1985. J. Immunol.    135:646-652.-   11. Mule, J. J., J. Yang, S. Shu, and S. A. Rosenberg. 1986. J.    Immunol. 136:3899-3909.-   12. Thompson, J. A., D. J. Peace, J. P. Klarnet, D. E. Kern, P. D.    Greenberg, and M. A. Cheever. 1986. J. Immunol. 137:3675-3680.-   13. Mazumder, A., T. J. Eberlein, E. A. Grimm, D. J. Wilson, A. M.    Keenan, R. Aamodt, and S. A. Rosenberg. 1984. Cancer 53:896-905.-   14. Rosenberg, S. A., M. T. Lotze, L. M. Muul, S. Leitman, A. E.    Chang, S. E. Ettinghausen, Y. L. Matory, J. M. Skibber, E.    Shiloni, J. T. Vetto, C. A. Seipp, C. Simpson, and C. M.    Reichert. 1985. N. Engl. J. Med. 313:1485-1492.-   15. Rosenberg, S. A., M. T. Lotze, J. C. Yang, P. M.    Aebersold, W. M. Linehan, C. A. Seipp, and D. E. White. 1989. Ann.    Surg. 210:474-485.-   16. Aebersold, P., C. Hyatt, S. Johnson, K. Hines, L. Korack, M.    Sanders, M. T. Lotze, S. Topalian, J. Yang, and S. A.    Rosenberg. 1991. J. Natl. Cancer Inst. 83:932-937.-   17. Fisher, R. I., C. A. J. Coltman, J. H. Doroshow, A. A.    Rayner, M. J. Hawkins, J. W. Miar, P. Wiernik, J. D. McMannis, G. R.    Weiss, K. A. Margolin, B. T. Gemlo, D. F. Hoth, D. R. Parkinson,    and E. Paietta. 1988. Ann. Intern. Med. 108:518-523.-   18. Thompson, J. A., K. L. Shulman, M. C. Benyunes, C. G.    Lindgren, C. Collins, P. H. Lange, W. H. Bush, Jr., L. A. Benz,    and A. Fefer. 1992. J. Clin. Oncol. 10:960-968.-   19. Rosenberg, S. A., P. Spiess, and R. Lafreniere. 1986. Science    233:1318-1321.-   20. Griffith, K. D., E. J. Read, and C. S. Carrasquillo. 1989. J.    Natl. Cancer Inst. 81:1709-1717.-   21. Taneja, S. S., W. Pierce, R. Figlin, and A. Belldegrun. 1995.    Urology 45:911-924.-   22. Topalian, S. L., D. Solomon, F. P. Avis, A. E. Chang, D. L.    Freerksen, W. M. Linehan, M. T. Lotze, C. N. Robertson, C. A.    Seipp, P. Simon, C. G. Simpson, and S. A. Rosenberg. 1988. J. Clin.    Oncol. 6:839-853.-   23. Rosenberg, S. A., B. S. Packard, P. M. Aebersold, D.    Solomon, S. L. Topalian, S. T. Toy, P. Simon, M. T. Lotze, J. C.    Yang, C. A. Seipp, C. G. Simpson, C. Carter, S. Bock, D.    Schwartzentruber, J. P. Wei, and D. E. White. 1988. N. Engl. J. Med.    319:1676-1680.-   24. Rosenberg, S. A., P. Aebersold, K. Cometta, A. Kasid, R. A.    Morgan, R. Moen, E. M. Karson, M. T. Lotze, J. C. Yang, S. L.    Topalian, M. J. Merino, K. Culver, A. D. Miller, R. M. Blaese,    and W. F. Anderson. 1990. N. Engl. J. Med. 323:570-578.-   25. Goedegebuure, P. S., L. M. Douville, H. Li, G. C.    Richmond, D. D. Schoof, M. Scavone, and T. J. Eberlein. 1995. J.    Clin. Oncol. 13:1939-1949.-   26. Peace, D. J. and M. A. Cheever. 1989. J. Exp. Med. 169:161-173.-   27. Lotze, M. T., Y. L. Matory, S. E. Ettinghausen, A. A.    Rayner, S. O. Sharrow, C. A. Seipp, M. C. Custer, and S. A.    Rosenberg. 1985. J. Immunol. 135:2865-2875.-   28. Higuchi, C. M., J. A. Thompson, F. B. Petersen, C. D. Buckner,    and A. Fefer. 1991. Blood 77:2561-2568.-   29. Rosenberg, S. A. 1984. J. Biol. Response Mod. 3:501-511.-   30. Ikarashi, H., K. Fujita, K. Takakuwa, S. Kodama, A. Tokunaga, T.    Takahashi, and K. Tanaka. 1994. Cancer Res. 54:190-196.-   31. Ikarashi, H., F. Fujita, S. Kodama, K. Tanaka, A. Tokunaga,    and T. Takahashi. 1995. Jpn. J. Cancer Res. 83:1359-1992.-   32. 1992. Long-term results of a randomized trial comparing    cisplatin with cisplatin and cyclophosphamide with cisplatin,    cyclophosphamide, and adriamycin in advanced ovarian cancer. GICOG    (Gruppo Interregionale Cooperativo Oncologico Ginecologia), Italy.    Gynecol. Oncol. 45:115-117.-   33. Yoshizawa, H., A. E. Chang, and S. Shu. 1991. J. Immunol.    147:729-737.-   34. Chang, A. E. and S. Shu. 1996. Crit. Rev. Oncol. Hematol.    22:213-228.-   35. Chang, A. E., A. Aruga, M. J. Cameron, V. K. Sondak, D. P.    Normolle, B. A. Fox, and S. Shu. 1997. J. Clin. Oncol. 15:796-807.-   36. Riddell, S. R. and P. D. Greenberg. 1990. J. Immunol. Methods    128:189-201.-   37. Melief, C. J. 1993. Semin. Hematol. 30:32-33.-   38. Schultze, J. L., S. Michalak, M. J. Seamon, G. Dranoff, K.    Jung, J. Daley, J. C. Delgado, J. G. Gribben, and L. M.    Nadler. 1997. J. Clin. Invest. 100:2757-2765.-   39. Schulz, M., P. Aichele, R. Schneider, T. H. Hansen, R. M.    Zinkernagel, and H. Hengartner. 1991. Eur. J. Immunol. 21:1181-1185.-   40. Riddell, S. R. and P. D. Greenberg. 1995. Cancer Treat. Res.    76:337-369.-   41. Riddell, S. R., K. S. Watanabe, J. M. Goodrich, C. R. Li, M. E.    Agha, and P. D. Greenberg. 1992. Science 257:238-241.-   42. Gedde-Dahl, T., III, B. Fossum, J. A. Eriksen, E. Thorsby,    and G. Gaudernack. 1993. Eur. J. Tmmunol. 23:754-760.-   43. Schlichtholz, B., Y. Legros, d. Gillet, C. Gaillard, M.    Marty, D. Lane, F. Calvo, and T. Soussi. 1992. Cancer Res.    52:6380-6384.-   44. Houbiers, J. G., H. W. Nijman, S. H. van der Burg, J. W.    Drijfhout, P. Kenemans, C. J. van de Velde, A. Brand, F.    Momburg, W. M. Kast, and C. J. Melief. 1993. Eur. J. Immunol.    23:2072-2077.-   45. Noguchi, Y., Y.-T. Chen, and L. J. Old. 1994. Proc. Natl. Acad.    Sci .USA 91:3171-3175.-   46. Yanuck, M., D. P. Carbone, C. D. Pendleton, I. Tsukui, S. F.    Winter, J. D. Minna, and J. A. Berzofsky. 1993. Cancer Res.    53:3257-2361.-   47. Nijman, H. W., J. G. Houbiers, S. H. van der Burg, M. P.    Vierboom, P. Kenemans, W. M. Kast, and C. J. Melief. 1993. J.    Immunother. 14:121-126.-   48. Smith, C. A., C. Y. C. Ng, H. E. Heslop, M. S. Holladay, S.    Richardson, E. V. Turner, S. K. Loftin, C. Li, and M. K.    Brenner. 1995. J. Hematother. 4:73-79.-   49. Papadopoulos, E. B., M. Ladanyi, D. Emanuel, S. Mackinnon, F.    Boulad, M. H. Carabasi, H. Castro-Malaspina, B. H. Childs, A. P.    Gillio, T. N. Small, J. W. Young, N. A. Kernan, and R. J.    O'Reilly. 1994. N. Engl. J. Med. 330:1185-1191.-   50. Osband, M. E., P. T. Lavin, R. K. Babayan, S. Graham, D. L.    Lamm, B. Parker, I. S. Sawczuk, S. Ross, and R. J. Krane. 1990.    Lancet 335:994-998.-   51. Gold, J. E. and M. E. Osband. 1994. Clin. Immunol. Immunopathol.    71:325-332.-   52. Gold, J. E. and M. E. Osband. 1994. Eur. J. Cancer    30A:1871-1882.-   53. Lavin, P. T., R. Maar, M. Franklin, S. Ross, J. Martin,    and M. E. Osband. 1992. Transplant. Proc. 24:3059-3064.-   54. Van Wauwe, J. P., J. R. De Mey, and J. G. Gooseens. 1980. J.    Immunol. 124:2708-2713.-   55. Meuer, S.C., J. C. Hodgdon, R. E. Hussey, J. P. Protentis, S. F.    Schlossman, and E. L. Reinherz. 1983. J. Exp. Med. 158:988-993.-   56. Meuer, S. C., R. E. Hussey, D. A. Cantrell, J. C. Hodgdon, S. F.    Schlossman, K. A. Smith, and E. L. Reinherz. 1984. Proc. Natl. Acad.    Sci. USA 81:1509-1513.-   57. Weiss, A. and J. B. Imboden. 1987. Adv. Immunol. 41:1-38.-   58. Loeffler, C. M., J. L. Platt, P. M. Anderson, E. Katsanis, J. B.    Ochoa, W. J. Urba, D. L. Longo, A. S. Leonard, and A. C.    Ochoa. 1991. Cancer Res. 51:2127-2132.-   59. Murphy, W. J., K. C. Conlon, T. J. Sayers, R. H. Wiltrout, T. C.    Back, J. R. Ortaldo, and D. L. Longo. 1993. J. Immunol.    150:3634-3642.-   60. Yun, Y. S., M. E. Hargrove, and C. Ting. 1989. Cancer Res.    49:4770-4774.-   61. Katsanis, E., Z. Xu, P. M. Anderson, B. B. Dancisak, M. A.    Bausero, D. J. Weisdorf, B. R. Blazar, and A. C. Ochoa. 1994. Bone    Marrow Transplant. 14:563-572.-   62. Ochoa, A. C., G. Gromo, B. J. Alter, P. M. Sondel, and F. H.    Bach. 1987. J. Immunol. 138:2728-2733.-   63. Anderson, P. M., F. H. Bach, and A. C. Ochoa. 1988. Cancer    Immunol. Immunother. 27:82-88.-   64. Chen, B. P., M. Malkovsky, J. A. Hank, and P. M. Sondel. 1987.    Cell Immunol. 110:282-293.-   65. Lotzova, E., C. A. Savary, R. B. Herberman, K. B.    McCredie, M. J. Keating, and E. J. Freireich. 1987. Nat. Immun. Cell    Growth Regul. 6:219-223.-   66. Yang, S.C., K. D. Fry, E. A. Grimm, and J. A. Roth. 1990. Arch.    Surg. 125:220-225.-   67. Ueda, M., I. D. Joshi, M. Dan, J. P. Uberti, T.-H. Chou, L. L.    Sensenbrenner, and L. G. Lum. 1993. Transplantation 56:351-356.-   68. Uberti, J. P., I. Joshi, M. Ueda, F. Martilotti, L. L.    Sensenbrenner, and L. G. Lum. 1994. Clin. Immunol. Immunopathol.    70:234-240.-   69. Anderson, P. M., B. R. Blazar, F. H. Bach, and A. C.    Ochoa. 1989. J. Immunol. 142:1383-1394.-   70. Anderson, P. M., A. C. Ochoa, N. K. C. Ramsay, D. Hasz, and D.    Weisdorf. 1992. Blood 80:1846-8153.-   71. Ting, C.-C., M. E. Hargrove, and Y. S. Yun. 1988. J. Immunol.    141:741-748.-   72. Ochoa, A. C., D. E. Hasz, R. Rezonzew, P. M. Anderson, and F. H.    Bach. 1989. Cancer Res. 49:963-968.-   73. Sosman, J. A., K. R. Oettel, J. A. Hank, P. Fisch, and P. M.    Sondel. 1989. Transplantation 48:486.-   74. Sosman, J. A., Oettel, K. R., Hank, J. A., and Sondel, P. M.    FASEB Journal 3(Pt 1), A506. 1989. Ref Type: Abstract-   75. Massaia, M., C. Attisano, S. Peola, L. Montacchini, P. Omede, P.    Corradini, D. Ferrero, M. Boccadoro, A. Bianchi, and A.    Pileri. 1993. Blood 82:1787-1797.-   76. Curti, B. C., D. L. Longo, A. C. Ochoa, K. C. Conlon, J. W.    Smith, II, W. G. Alvord, S. P. Creekmore, R. G. Fenton, B. L.    Gause, J. Holmlund, J. E. Janik, J. Ochoa, P. A. Rice, W. H.    Sharfman, M. Sznol, and W. J. Urba. 1993. J. Clin. Oncol.    11:652-660.-   77. Saxton, M. L., D. L. Longo, H. E. Wetzel, H. Tribble, W. G.    Alvord, L. W. Kwak, A. S. Leonard, C. D. Ullmann, B. D. Curti,    and A. C. Ochoa. 1997. Blood 89:2529-2536.-   78. Curti, B. D., A. C. Ochoa, G. C. Powers, W. C. Kopp, W. G.    Alvord, J. E. Janik, B. L. Gause, B. Dunn, M. S. Kopreski, R.    Fenton, A. Zea, C. Dansky-Ullmann, S. Strobl, L. Harvey, E.    Nelson, M. Sznol, and D. L. Longo. 1998. J. Clin. Oncol.    16:2752-2760.-   79. June, C. H., J. A. Ledbetter, P. S. Linsley, and C. B.    Thompson. 1990. Immunol. Today 11:211-216.-   80. Costello, R., C. Cerdan, C. Pavon, H. Brailly, C. Hurpin, C.    Mawas, and D. Olive. 1993. Eur. J. Immunol. 23:608-613.-   81. June, C. H., J. A. Bluestone, L. M. Nadler, and C. B.    Thompson. 1994. Immunol. Today 15:321-331.-   82. Jenkins, M. K. and J. G. Johnson. 1993. Curr. Opin. Immunol.    5:361-367.-   83. Schwartz, R. H. 1992. Cell 71:1065-1068.-   84. Thompson, C. B., T. Lindsten, J. A. Ledbetter, S. L.    Kunkel, H. A. Young, S. G. Emerson, J. M. Leiden, and C. H.    June. 1989. Proc. Natl. Acad. Sci. USA 86:1333-1337.-   85. Rosenberg, E. S., J. M. Billingsley, A. M. Caliendo, S. L.    Boswell, P. E. Sax, S. A. Kalams, and B. D. Walker. 1997. Science    278:1447-1450.-   86. Garlie, N. K., A. V. LeFever, R. E. Siebenlist, B. L.    Levine, C. H. June, and L. G. Lum. 1999. J. Immunother. 4:335-345.-   87. Levine, B. L., Y. Ueda, N. Craighead, M. L. Huang, and C. H.    June. 1995. Int. Immunol. 7:891-904.-   88. Boise, L. H., A. J. Minn, M. A. Accavitti, C. H. June, T.    Lindsten, and C. B. Thompson. 1995. Immunity 3:87-98.-   89. Boise, L. H., P. J. Noel, and C. B. Thompson. 1995. Curr. Opin.    Immunol. 7:620-625.-   90. Harada, M., T. Okamoto, K. Omoto, K. Tamada, M. Takenoyama, C.    Hirashima, O. Ito, G. Kimura, and K. Nomoto. 1996. Immunology    87:447-453.-   91. Shibuya, T. H., Wei, W. Z., Johnson, R. D., Zormeier, M.,    Meleca, R. H., Mathog, R. H., June, C. H., and Lum, L. G.    Anti-CD3/CD28 bead costimulation overcomes regional    immunosuppression in HNSCC (head and neck squamous cell carcinoma)    patients. ARO Abstracts (Assn for Reseach in Otolaryngology],    #659. 1998. Ref Type: Abstract-   92. Cayota, A., F. Vuillier, J. Siciliano, and G. Dighiero. 1994.    Int. Immunol. 6:611-621.-   93. Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet,    and J. P. Allison. 1992. Nature 356:607-609.-   94. Guinan, E. C., J. G. Gribben, V. A. Boussiotis, G. J. Freeman,    and L. M. Nadler. 1994. Blood 84:3261-3282.-   95. Lum, L. G., LeFever, A. V., Treisman, J. S., Hanson, J. P., Jr.,    Garlie, N. K., Kistler, A. M., Yuille, D. L., Levine, B. L., and    June, C. H. Phase I study of anti-CD3/anti-CD23 coactivated T cells    (COACTS) in cancer patients: enhanced TH1 responses in vivo.    Experimental Hematology 26, 772. 1998. Ref Type: Abstract-   96. Renner, C. and M. Pfreundschuh. 1995. Immunol. Rev. 145:179-209.-   97. Raso, V. and T. Griffin. 1981. Cancer Res. 41:2073-2078.-   98. Titus, J. A., P. Perez, A. Kaubisch, M. A. Garrido, and D. M.    Segal. 1987. J. Immunol. 139:3153-3158.-   99. Perez, P., J. A. Titus, M. T. Lotze, F. Cuttitta, D. L.    Longo, E. S. Groves, H. Rabin, P. J. Durda, and D. M.    Segal. 1986. J. Immunol. 137:2069-2072.-   100. Segal, D. M., M. A. Garrido, P. Perez, J. A. Titus, D. A.    Winkler, D. B. Ring, A. Kaubisch, and J. R. Wunderlich. 1988. Mol.    Immunol. 25:1099-1103.-   101. Bonino, L. D., L. B. De Monte, G. C. Sapnoli, R. Vola, M.    Mariani, D. Barone, A. M. Moro, P. Riva, M. R. Niotra, P. G. Natali,    and F. Malavasi. 1995. Int. J. Cancer 61:509-515.-   102. Kaneko, T., Y. Fusauchi, Y. Kakui, M. Masuda, M. Akahoshi, M.    Termura, T. Motoji, K. Okumura, H. Mizoguchi, and K. Oshimi. 1993.    Blood 81:1333-1341.-   103. Katayose, Y., T. Kudo, M. Suzuki, M. Shinoda, S. Saijyo, N.    Sakurai, H. Saeki, K. Fukuhara, K. Imai, and S. Matsuno. 1996.    Cancer Res. 56:4205-4212.-   104. Mack, M., R. Gruber, S. Schmidt, G. Riethmtiller, and P.    Kufer. 1997. J. Immunol. 158:3965-3970.-   105. Lamers, C. H. J., R. J. van de Griend, E. Braakman, C. P. M.    Ronteltap, J. Bénard, and G. Stoter. 1992. Int. J. Cancer    51:973-979.-   106. Canevari, S., D. Mezzanzanica, A. Mazzoni, D. R. M. Negri, V.    Ramakrishna, R. L. H. Bohuis, M. I. Colnaghi, and G. Bolis. 1995.    J.Hematother. 4:423-427.-   107. Canevari, S., G. Stoter, F. Arienti, G. Bolis, M. I.    Colnaghi, E. M. Di Re, A. M. M. Eggermont, S. H. Goey, J. W.    Gratama, C. H. J. Lamers, M. A. Nooy, G. Parmiani, F.    Raspagliesi, F. Ravagnani, G. Scarfone, J. B. Trimbos, S. O.    Warnaar, and R. L. H. Bolhuis. 1995. J. Natl. Cancer Inst.    87:1463-1469.-   108. Bolhuis, R. L. H., C. H. J. Lamers, S. H. Goey, A. M. M.    Eggermont, J. B. M. Z. Trimbos, G. Stoter, A. Lanzavecchia, E. di    Re, S. Mioth, F. Raspagliesi, L. Rivoltini, and M. I.    Colnaghi. 1992. Int. J. Cancer78-81.-   109. Haas, C., G. Strauss, G. Moldenhauer, R. M. Iorio, and V.    Schirrmacher. 1998. Clin. Cancer Res. 4:721-730.-   110. Zhu, Z., T. Ghose, S. H. Lee, L. A. Fernandez, L. A.    Kerr, J. H. Donohue, and D. J. McKean. 1994. Cancer Lett.    86:127-134.-   111. Van Dijk, J., S. O. Warnaar, J. D. van Eendenburg, M.    Thienpont, E. Braakman, J. H. Boot, G. J. Fleuren, and R. L.    Bolhuis. 1989. Int. J. Cancer 43:344-349.-   112. Tahara, H. and M. T. Lotze. 1995. Gene Ther. 2:96-106.-   113. Kroesen, B. J., A. ter Haar, P. Willemse, D. T.    Sleijfer, E. G. E. de Vries, N.H. Mulder, H. H. Berendsen, P. C.    Limburg, H. T. The, and L. de Leij. 1993. Cancer Immunol.    Immunother. 37:401-407.-   114. Demanet, C., J. Brissinck, J. De Jong, and K. Thielemans. 1996.    Blood 87:4390-4398.-   115. Bohlen, H., T. Hopff, O. Manzke, A. Engert, D. Kube, P. D.    Wickramanayake, V. Diehl, and H. Tesch. 1993. Blood 82:1803-1812.-   116. Bohlen, H., O. Manzke, B. Patel, G. Moldenhauer, B. DOrken, V.    von Fliedner, V. Diehl, and H. Tesch. 1993. Cancer Res.    43:4310-4314.-   117. Anderson, P. M., W. Crist, D. Hasz, A. J. Carroll, D. E. Myers,    and F. M. Uckun. 1992. Blood 80:2826-2834.-   118. Bejeck, B. E., D. Wang, E. Berven, C. A. Pennell, S.C.    Peiper, S. Poppema, F. M. Uckun, and J. H. Kersey. 1995. Cancer Res.    55:2346-2351.-   119. de Gast, G. C., I.-A. Haagen, A. A. van Houten, S.C.    Klein, A. J. Duits, R. A. de Weger, T. M. Vroom, M. R. Clark, J.    Phillips, A. J. G. van Dijk, W. B. M. de Lau, and B. J. E. G.    Bast. 1995. Cancer Immunol. Immunother. 40:390-396.-   120. Klein, S.C., L. H. Boer, R. A. de Weger, G. C. de Gast,    and E. J. E. G. Bast. 1997. Scand. J. Immunol. 46:452-458.-   121. Chapoval, A. I., H. Nelson, and C. Thibault. 1995. J. Immunol.    155:1296-1303.-   122. Kuwahara, M., M. Kuroki, F. Arakawa, T. Senba, Y. Matsuoka, T.    Hideshima, Y. Yamashita, and H. Kanda. 1997. Anticancer Res.    16:2661-2668.-   123. Brossart, P., G. Stuhler, T. Flad, S. Stevanovic, H.-G.    Rammensee, L. Kanz, and W. Brugger. 1998. Cancer Res. 58:732-736.-   124. Renner, C., W. Jung, U. Sahin, R. Denfeld, C. Pohl, L.    Triimper, F. Hartmann, V. Diehl, R. van Lier, and M.    Pfreundschuh. 1994. Science 264:833-835.-   125. Renner, C., S. Bauer, U. Sahin, W. Jung, R. van Lier, G.    Jacobs, G. Held, and M. Pfreundschuh. 1996. Blood 87:2930-2937.-   126. Pohl, C., R. Denfeld, C. Renner, W. Jung, H. Bohlen, U.    Sahin, A. Hombach, R. van Lier, M. Schwonzen, V. Diehl, and M.    Pfreundschuh. 1993. Int. J. Cancer 54:820-827.-   127. Hayden, M. S., P. S. Linsley, M. A. Gayle, J. Bajorath, W. A.    Brady, N. A. Norris, H. P. Fell, J. A. Ledbetter, and L. K.    Gilliland. 1994. Therapeut. Immunol. 1:3-15.-   128. Alvarez-Vallina, L. and R. E. Hawkins. 1996. Eur. J. Immunol.    26:2304-2309.-   129. Renner, C., W. Jung, U. Sahin, R. van Lier, and M.    Pfreundschuh. 1995. Eur. J. Immunol. 25:2027-2033.-   130. Mazzoni, A., D. Mezzanzanica, G. Jung, H. Wolf, M. I. Colnaghi,    and S. Canevari. 1996. Cancer Res. 56:5443-5449.-   131. Hombach, A., T. Tillmann, M. Jensen, C. Heuser, R. Sircar, V.    Diehl, W. Kruis, and C. Pohl. 1997. Clin. Exp. Immunol. 108:352-357.-   132. Hayden, M. S., L. S. Grosmaire, N. A. Norris, L. K.    Gilliland, G. Winberg, D. Tritschler, T. T. Tsu, P. S.    Linsley, R. S. Mittler, P. D. Senter, H. P. Fell, and J. A.    Ledbetter. 1996. Tissue Antigens 48:242-254.-   133. Michon, J., S. Moutel, J. Barbet, J.-L. Romet-Lemonne, Y. M.    Deo, W. H. Fridman, and J.-L. Tcillaud. 1995. Blood 86:1124-1130.-   134. Weiner, L. M., J. I. Clark, M. Davey, W. S. Li, I. G. de    Palazzo, D. B. Ring, and R. K. Alpaugh. 1995. Cancer Res.    55:4586-4593.-   135. Valone, F. H., R A. Kaufman, P. M. Guyre, L. D. Lewis, V.    Memoli, Y. Deo, R. Graziano, J. L. Fisher, L. Meyer, and M.    Mrozek-Orlowski. 1995. J. Clin. Oncol. 13:2281-2292.-   136. James, N., P. Atherton, A. Koletsky, N. Tchekmedyian, and R.    Curnow. 1998. Proc. Am. Soc. Clin. Oncol. 17:436a.-   137. Shalaby, M. R., H. M. Shepard, L. Presta, M. L.    Rodrigues, P. C. L. Beverley, M. Feldmann, and P. Carter. 1992. J.    Exp. Med. 175:217-225.-   138. Zhu, Z., G. D. Lewis, and P. Carter. 1995. Int. J. Cancer    62:319-324.-   139. Nakamura, Y., Y. Tokuda, M. Iwasawa, H. Tsukamoto, M.    Kidokoro, N. Kobayashi, S. Kato, T. Mitomi, S. Habu, and T.    Nishimura. 1992. [Comment in: Br.J.Cancer 1993; 67:865-7]. Br. J.    Cancer 66:20-26.-   140. Dawson, N, Moul, J., and Higano, C. Hormone-refractory prostate    cancer: Current issues and treatment options. 1-8. 1999. Physicians    & Scientists Publishing Co., Inc. Ref Type: Pamphlet-   141. Waselenko, J. K. and N. A. Dawson. 1997. Oncology 11:1551-1567.-   142. Watanabe, M., T. Nakada, and H. Yuta. 1999. Int. Urol. Nephrol.    31:61-73.-   143. Schwartz, S. Jr., Caceres, C., Morote, J., de Tones, I.,    Rodriguez-Vallejo, J. M., Gonzalez, J., and Reventos, J. Int. J.    Oncol. 14(2), 367-371. 1999. Ref Type: Journal (Full)-   144. Morote, J., I. de Tones, C. Caceres, C. Vallejo, S. Jr.    Schwartz, and J. Reventos. 1999. Int. J. Cancer 84:421-425.-   145. Ullrich, A. and J. Schlessinger. 1990. Cell 61:203-212.-   146. Disis, M. L. and M. A. Cheever. 1997. Adv. Cancer Res.    71:343-371.-   147. Mark, D. F., L. Feldman, S. Das, H. Kye, S. Mark, C. L. Sun,    and M. Samy. 1999. Exp. Mol. Pathol 66:170-178.-   148. Bubendorf, L., J. Kononen, P. Koivisto, P. Schraml, H.    Moch, T. C. Gasser, N. Willi, G. Sauter, and O. P.    Kallioniemi. 1999. Cancer Res. 59:803-806.-   149. Eshhar, Z., T. Waks, G. Gross, and D. G. Schindler. 1993. Proc.    Natl. Acad. Sci. USA 90:720-724.-   150. Hwu, P., G. E. Shafer, J. S. Treisman, D. G. Schindler, G.    Gross, R. Cowherd, S. A. Rosenberg, and Z. Eshhar. 1993. J. Exp.    Med. 178:361-366.-   151. Hwu, P., J. C. Yang, R. Cowherd, J. S. Treisman, G. E.    Shafer, Z. Eshhar, and S. A. Rosenberg. 1995. Cancer Res.    55:3369-3373.-   152. Eshhar, Z., N. Bach, C. J. Fitzer-Attas, G. Gross, J.    Lustgarten, T. Waks, and D. G. Schindler. 1996. Springer Semin.    Immunopathol. 18:199-209.-   153. Altenschmidt, U., D. Moritz, and B. Groner. 1997. J. Mol. Med.    75:259-266.-   154. Fitzer-Attas, C. J. and Z. Eshhar. 1998. Adv. Drug Delivery    Rev. 31:171-182.-   155. Plavec, I., M. Agarwal, K. E. Ho, M. Pineda, J. Auten, J.    Baker, H. Matsuzaki, S. Escaich, M. Bonyhadi, and E. Bohnlein. 1997.    Gene Ther. 4:128-139.-   156. Quinn, E. R., L. G. Lum, and K. T. Trevor. 1998. Hum. Gene    Ther. 9:1457-1467.-   157. Trevor, K. T., E. R. Quinn, M. Sen, D. Wankowski, K. Knox,    and L. G. Lum. 2000. Bispecific antibody reactivation of    gene-transduced T cells: Implications for cancer immunotherapy and    gene therapy. Tumor Targeting (In press).-   158. Stockmeyer, B., T. Valerius, R. Repp, I. A. F. M. Heijnen,    H.-J. Biihring, Y. M. Deo, J. R. Kalden, M. Gramatzki, and J. G. J.    van de Winkel. 1997. Cancer Res. 57:696-701.-   159. Baselga, J., L. Norton, J. Albanell, Y. M. Kim, and J.    Mendelsohn. 1998. Cancer Res. 58:2825-2831.-   160. Jones, R. J., G. B. Vogelsang, A. D. Hess, E. R. Farmer, R. B.    Maim, R. B. Geller, S. Piantadosi, and G. W. Santos. 1989. Lancet    1:754-757.-   161. Baselga, J., D. Tripathy, J. Mendelsohn, S. A. Baughman, C. C.    Benz, L. Dantis, N. T. Sklarin, A. D. Seidman, C. A. Hudis, J.    Moore, P. P. Rosen, T. Twaddell, I. C. Henderson, and L.    Norton. 1996. J. Clin. Oncol. 14:737-744.-   162. Lum, L. G., Treisman, J. S., Taylor, R. F., and LeFever, A. V.    Phase I/II trial of activated T cells (ATC), IL-2, and GM-CSF after    PBSC transplant for stage IIIb or IV breast cancer. Blood 90(Suppl),    381b. 1997. Ref Type: Abstract-   163. Thakur, M. L., R. E. Coleman, and M. J. Welch. 1977. J. Lab.    Clin. Med. 89:217-228.-   164. Beightol, R. W. and W. J. Baker. 1980. Am. J. Hosp. Pharm.    37:847-850.

1. A method for treatment of a patient suffering from cancer, saidmethod comprising the steps of: (a) isolating peripheral bloodmononuclear cells comprising T cells; (b) activating the T cells by exvivo stimulation, and expanding said activated T cells; (c) arming ofactivated T cells with bispecific antibodies capable of binding to the Tcell receptor complex of a T cell and/or Fc-receptor positive cells, andto tumor-associated antigens on a tumor cell, wherein the bispecificantibodies redirect the activated T cells to kill the tumor cells bybinding to the tumor cells and triggering the secretion of tumoricidalcytokines and chemokines; and, (d) infusing a composition comprising thearmed, activated T cells into the patient in a therapeutically effectiveamount able to (i) target and contact multiple tumor cells with thearmed activated T cells to which the bispecific antibody remains boundand (ii) kill the tumor cells by secreting cytokines.
 2. The method ofclaim 1, wherein said method further comprises co-infusing intravenouslyor co-injecting into a tumor arterial supply or tumor site a compositionof dendritic cells with the armed, activated T cells.
 3. (canceled) 4.The method of claim 2, wherein the armed, activated T cells arecoinfused with IL-2, IL-12, GM-CSF or other immune augmenting cytokines.5. (canceled)
 6. The method according to claim 1, wherein the bispecificantibody is comprised of two monoclonal antibodies.
 7. The methodaccording to claim 6, wherein each of the specificities of saidbispecific antibody are directed to a tumor antigen and the T cellreceptor complex.
 8. The method according to claim 6, wherein themonoclonal antibodies are chemically heteroconjugated to form thebispecific antibody.
 9. The method according to claim 1, wherein thebispecific antibody is comprised of monoclonal antibodies directed toany tumor associated antigen.
 10. The method according to claim 1,wherein the anti-T cell receptor monoclonal antibody component of saidbispecific antibody is directed against CD3 of the T cell receptorcomplex.
 11. The method according to claim 1, wherein the Tcell-mediated cytotoxicity of a tumor occurs in immunosuppressedpatients.
 12. The method according to claim 1, wherein T cell-mediatedcytotoxicity occurs in patients susceptible to, or suffering fromdiseases associated with abnormal cellular proliferation or growth. 13.The method according to claim 1, wherein the armed T cell can be frozenand thawed for use in a patient in need of such therapy.
 14. The methodaccording to claim 1, wherein the T cell is armed with a bispecificantibody dose of between about 0.5 ng per million T cells to betweenabout 500 ng per million T cells.
 15. The method of claim 14, whereinthe arming dose is optimized for each individual patient by titrating athawed aliquot of the patients activated T cells to achieve a percentspecific cytotoxicity level at an effector to target ratio from betweenabout 25:1 to at least about 30% against a tumor target.
 16. The methodof claim 15, wherein the infusing dose is at least about 2 billion armedT cells.
 17. The method of claim 15, wherein the patient receives atleast about four infusions.
 18. The method according to claim 1, whereinthe T cell is CD3/CD8 positive.
 19. The method according to claim 1,wherein the T cell is a CD3/CD4 positive cell.
 20. The method accordingto claim 1, wherein the armed T cells from a patient can beco-administered with other forms of therapy and/or immunocompetent naveT cells, and immunocompetent nave B cells. 21-89. (canceled)
 90. Amethod for treatment of a patient suffering from cancer, said methodcomprising the steps of: (a) isolating peripheral blood mononuclearcells comprising T cells; (b) activating of the T cells by ex vivostimulation and expanding of said activated T cells; (c) arming ofactivated T cells with bispecific antibodies capable of binding to the Tcell receptor complex of a T cell and/or Fc-receptor positive cells, andto tumor-associated antigens on a tumor cell, wherein the bispecificantibodies redirect the activated T cells to kill the tumor cells bybinding to the tumor cells and triggering the secretion of tumoricidalcytokines and chemokines; and, (d) infusing a composition comprising thearmed, activated T cells into the patient in a therapeutically effectiveamount able to (i) target and contact multiple tumor cells with thearmed activated T cells to which the bispecific antibody remains boundand (ii) kill the tumor cells by secreting cytokines.
 91. The method ofclaim 90, wherein the armed, activated T cells are coinfused with IL-2,IL-12, GM-CSF or other immune augmenting cytokines.
 92. The methodaccording to claims 91, wherein the bispecific antibody is comprised oftwo monoclonal antibodies.
 93. The method according to claim 92, whereineach of the specificities of said bispecific antibody are directed to atumor antigen and the T cell receptor complex.
 94. The method of claim90, wherein the autologous cells induce the proliferation of T cellsspecific for different epitopes on the tumor cell.
 95. The method ofclaim 90, wherein the bispecific antibody remains bound to the armed Tcell allowing the armed T cell to target and kill multiple target cellswhich the armed T cell recognizes the desired antigens on the targetcells. 96-105. (canceled)